Antisense compounds targeting leucine-rich repeat kinase 2 (LRRK2) for the treatment of parkinsons disease

ABSTRACT

The present disclosure relates generally to compounds comprising oligonucleotides complementary to a Leucine-Rich-Repeat-Kinase (LRRK2) RNA transcript. Certain such compounds are useful for hybridizing to a LRRK2 RNA transcript, including but not limited to a LRRK2 RNA transcript in a cell. In certain embodiments, such hybridization results in modulation of splicing of the LRRK2 transcript. In certain embodiments, such compounds are used to treat one or more symptoms associated with Parkinson&#39;s disease.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. application Ser. No.15/349,840, filed Nov. 11, 2016, now U.S. Pat. No. 9,840,710, issuedDec. 12, 2017, which is a non-provisional application of U.S.Provisional Application 62/257,109, filed Nov. 18, 2015, the disclosuresof each of which are incorporated by reference in their entirety.

SEQUENCE LISTING

The sequence listing submitted herewith is incorporated by reference inits entirety.

FIELD OF THE DISCLOSURE

The present disclosure relates generally to compounds comprisingoligonucleotides complementary to a Leucine-Rich-Repeat-Kinase (LRRK2)RNA transcript. Certain such compounds are useful for hybridizing to aLRRK2 transcript, including but not limited to a LRRK2 RNA transcript ina cell. In certain embodiments, such hybridization results in modulationof splicing of the LRRK2 transcript. In certain embodiments, suchcompounds are used to treat one or more symptoms associated withParkinson's Disease (PD).

BACKGROUND OF THE DISCLOSURE

Parkinson's disease belongs to a group of conditions called motor systemdisorders, which are the result of the loss of dopamine-producing braincells. The four primary symptoms of PD are tremor, or trembling inhands, arms, legs, jaw, and face; rigidity, or stiffness of the limbsand trunk; bradykinesia, or slowness of movement; and posturalinstability, or impaired balance and coordination. As these symptomsbecome more pronounced, patients may have difficulty walking, talking,or completing other simple tasks. Other symptoms may include depressionand other emotional changes; difficulty in swallowing, chewing, andspeaking; urinary problems or constipation; skin problems; and sleepdisruptions. There are currently no blood or laboratory tests that havebeen proven to help in diagnosing sporadic PD, and diagnosis is based onmedical history and a neurological examination.

Currently, there is no cure for PD, but a variety of medications providerelief from the symptoms. Usually, affected individuals are givenlevodopa combined with carbidopa, which delays the conversion oflevodopa into dopamine until it reaches the brain. Nerve cells can uselevodopa to make dopamine and replenish the brain's dwindling supply.Although levodopa helps at least three-quarters of parkinsonian cases,not all symptoms respond equally to the drug. Bradykinesia and rigidityrespond best, while tremor may be only marginally reduced. Problems withbalance and other symptoms may not be alleviated at all.Anticholinergics may help control tremor and rigidity. Other drugs, suchas bromocriptine, pramipexole, and ropinirole, mimic the role ofdopamine in the brain, causing the neurons to react as they would todopamine. An antiviral drug, amantadine, also appears to reducesymptoms.

An estimated 53 million people have PD resulting in about 103,000 deathsglobally. Parkinson's disease typically occurs in people over the age of60, with males being affected more often than females. The average lifeexpectancy following diagnosis is between 7 and 14 years. Parkinson'sdisease is typically idiopathic (having no specific known cause);however, a proportion of cases can be attributed to known geneticfactors. For example, mutations in specific genes have been shown tocause PD (e.g., alpha-synuclein (SNCA), parkin (PRKN), leucine-richrepeat kinase 2. (LRRK2), PTEN-induced putative kinase 1 (PINK1), DJ-1and ATP13A2). Mutations in LRRK2 are the most common known cause offamilial and sporadic PD, accounting for approximately 5% of individualswith a family history of the disease and 3% of sporadic cases. The LRRK2G2019S gain of function gene mutation is one of the most prevalentmutations contributing to PD pathogenesis. While treatments for PD areavailable, more effective therapies are needed.

SUMMARY OF THE DISCLOSURE

The present disclosure relates to general compounds and methods to treatParkinson's disease in subjects using antisense oligonucleotides (ASOs)that block specific pre-mRNA splicing events in LRRK2 gene transcriptsresulting in non-sense mRNAs or mRNAs that code for LRRK2 proteins withlower kinase activity.

In one aspect, the disclosure provides a compound comprising a modifiedoligonucleotide having 8 to 30 linked nucleosides having a nucleobasesequence comprising a complementary region, wherein the complementaryregion comprises at least 8 contiguous nucleobases complementary to anequal-length portion of a target region of a leucine-rich repeat kinase2 (LRRK2) transcript. In certain embodiments, the target region of theLRRK2 transcript comprises at least a portion of exon 2, exon 4, exon 31or exon 41 of the LRRK2 transcript. In an embodiment, the target regioncan be a splice site in the LRRK2 gene. In other embodiments, thenucleobase sequence of the antisense oligonucleotide comprises SEQ IDNO:01, SEQ ID NO:02, SEQ ID NO:06, SEQ ID NO:07, SEQ ID NO:08, or SEQ IDNO:09.

In another aspect, the disclosure provides a pharmaceutical compositioncomprising at least one compound as described herein and apharmaceutically acceptable carrier or diluent.

In yet another aspect, the disclosure provides a method of modulatingsplicing or expression of a LRRK2 transcript in a cell comprisingcontacting the cell with at least one compound as described herein.

The yet another aspect, the disclosure provides a method of treatingParkinson's disease, comprising administering at least one compound asdescribed herein to an animal in need thereof.

The present disclosure provides the following non-limiting numberedembodiments:

Embodiment 1

A compound comprising a modified oligonucleotide consisting of 8 to 30linked nucleosides and having a nucleobase sequence comprising acomplementary region, wherein the complementary region comprises atleast 8 contiguous nucleobases complementary to an equal-length portionof a target region of a leucine-rich repeat kinase 2 (LRRK2) transcript.

Embodiment 2

The compound of embodiment 1, wherein the target region of the LRRK2transcript comprises at least a portion of exon 2, exon 4, exon 31 orexon 41 of the LRRK2 transcript.

Embodiment 3a

The compound of embodiment 1, wherein the target region of the LRRK2transcript comprises at least a portion of exon 2 of the LRRK2transcript.

Embodiment 3b

The compound of embodiment 1, wherein the target region of the LRRK2transcript comprises at least a portion of exon 4 of the LRRK2transcript.

Embodiment 4

The compound of embodiment 1, wherein the target region of the LRRK2transcript comprises at least a portion of exon 31 of the LRRK2transcript.

Embodiment 5

The compound of embodiment 1, wherein the target region of the LRRK2transcript comprises at least a portion of exon 41 of the LRRK2transcript.

Embodiment 6

The compound of embodiment 1, wherein the target region of the LRRK2transcript comprises a splice site.

Embodiment 7

The compound of embodiment 1, wherein the LRRK2 transcript encodes aprotein that has a G2019S mutation or a R1441C mutation.

Embodiment 8

The compound of any of embodiments 1 to 7, wherein the complementaryregion of the modified oligonucleotide is at least 80%, at least 85%, atleast 90%, at least 95% or at least 100% complementary to the targetregion.

Embodiment 9

The compound of any of embodiments 1 to 8, wherein the complementaryregion of the modified oligonucleotide comprises at least 10 contiguousnucleobases.

Embodiment 10

The compound of any of embodiments 1 to 8, wherein the complementaryregion of the modified oligonucleotide comprises at least 12 contiguousnucleobases.

Embodiment 11

The compound of any of embodiments 1 to 8, wherein the complementaryregion of the modified oligonucleotide comprises at least 14 contiguousnucleobases.

Embodiment 12

The compound of any of embodiments 1 to 8, wherein the complementaryregion of the modified oligonucleotide comprises at least 15 contiguousnucleobases.

Embodiment 13

The compound of any of embodiments 1 to 8, wherein the complementaryregion of the modified oligonucleotide comprises at least 16 contiguousnucleobases.

Embodiment 14

The compound of any of embodiments 1 to 8, wherein the complementaryregion of the modified oligonucleotide comprises at least 17 contiguousnucleobases.

Embodiment 15

The compound of any of embodiments 1 to 8, wherein the complementaryregion of the modified oligonucleotide comprises at least 18 contiguousnucleobases.

Embodiment 16

The compound of any of embodiments 1 to 8, wherein the complementaryregion of the modified oligonucleotide comprises at least 19 contiguousnucleobases.

Embodiment 17

The compound of any of embodiments 1 to 8, wherein the complementaryregion of the modified oligonucleotide comprises at least 20 contiguousnucleobases.

Embodiment 18

The compound of any of embodiments 1 to 17, wherein the nucleobasesequence of the oligonucleotide is at least 80% complementary to anequal-length region of the LRRK2 transcript, as measured over the entirelength of the oligonucleotide.

Embodiment 19

The compound of any of embodiments 1 to 17, wherein the nucleobasesequence of the oligonucleotide is at least 90% complementary to anequal-length region of the LRRK2 transcript, as measured over the entirelength of the oligonucleotide.

Embodiment 20

The compound of any of embodiments 1 to 17, wherein the nucleobasesequence of the oligonucleotide is 100% complementary to an equal-lengthregion of the LRRK2 transcript, as measured over the entire length ofthe oligonucleotide.

Embodiment 21

The compound of any of embodiments 1 to 20, wherein the nucleobasesequence of the antisense oligonucleotide comprises any one of SEQ IDNO:01, SEQ ID NO:02, SEQ ID NO:06, SEQ ID NO:07, SEQ ID NO:08, or SEQ IDNO:09.

Embodiment 22

The compound of any of embodiments 1 to 21, wherein the modifiedoligonucleotide comprises at least one modified nucleoside.

Embodiment 23

The compound of embodiment 22, wherein at least one modified nucleosidecomprises a modified sugar moiety.

Embodiment 24

The compound of embodiment 23, wherein at least one modified sugarmoiety is a 2′-substituted sugar moiety.

Embodiment 25

The compound of embodiment 24, wherein the 2′-substitutent of at leastone 2′-substituted sugar moiety is selected from among: 2′-OMe, 2′-F,and 2′-MOE.

Embodiment 26

The compound of any of embodiments 22 to 25, wherein the 2′-substituentof at least one 2′-substituted sugar moiety is a 2′-MOE.

Embodiment 27

The compound of any of embodiments 1 to 26, wherein at least onemodified sugar moiety is a bicyclic sugar moiety.

Embodiment 28

The compound of embodiment 27, wherein at least one bicyclic sugarmoiety is LNA or cEt.

Embodiment 29

The compound of any of embodiments 1 to 28, wherein at least one sugarmoiety is a sugar surrogate.

Embodiment 30

The compound of embodiment 29, wherein at least one sugar surrogate is amorpholino.

Embodiment 31

The compound of embodiment 29, wherein at least one sugar surrogate is amodified morpholino.

Embodiment 32

The compound of any of embodiments 1 to 31, wherein the modifiedoligonucleotide comprises at least 5 modified nucleosides, eachindependently comprising a modified sugar moiety.

Embodiment 33

The compound of embodiment 32, wherein the modified oligonucleotidecomprises at least 10 modified nucleosides, each independentlycomprising a modified sugar moiety.

Embodiment 34

The compound of embodiment 32, wherein the modified oligonucleotidecomprises at least 15 modified nucleosides, each independentlycomprising a modified sugar moiety.

Embodiment 35

The compound of embodiment 32, wherein each nucleoside of the modifiedoligonucleotide is a modified nucleoside, each independently comprisinga modified sugar moiety

Embodiment 36

The compound of any of embodiments 1 to 35, wherein the modifiedoligonucleotide comprises at least two modified nucleosides comprisingmodified sugar moieties that are the same as one another.

Embodiment 37

The compound of any of embodiments 1 to 35, wherein the modifiedoligonucleotide comprises at least two modified nucleosides comprisingmodified sugar moieties that are different from one another.

Embodiment 38

The compound of any of embodiments 1 to 37, wherein the modifiedoligonucleotide comprises a modified region of at least 5 contiguousmodified nucleosides.

Embodiment 39

The compound of any of embodiments 1 to 38, wherein the modifiedoligonucleotide comprises a modified region of at least 10 contiguousmodified nucleosides.

Embodiment 40

The compound of any of embodiments 1 to 39, wherein the modifiedoligonucleotide comprises a modified region of at least 15 contiguousmodified nucleosides.

Embodiment 41

The compound of any of embodiments 1 to 39, wherein the modifiedoligonucleotide comprises a modified region of at least 20 contiguousmodified nucleosides.

Embodiment 42

The compound of any of embodiments 36 to 41, wherein each modifiednucleoside of the modified region has a modified sugar moietyindependently selected from among: 2′-F, 2′-OMe, 2′-MOE, cEt, LNA,morpholino, and modified morpholino.

Embodiment 43

The compound of any of embodiments 36 to 42 wherein the modifiednucleosides of the modified region each comprise the same modificationas one another.

Embodiment 44

The compound of embodiment 43, wherein the modified nucleosides of themodified region each comprise the same 2′-substituted sugar moiety.

Embodiment 45

The compound of embodiment 43, wherein the 2′-substituted sugar moietyof the modified nucleosides of the region of modified nucleosides isselected from 2′-F, 2′-OMe, and 2′-MOE.

Embodiment 46

The compound of embodiment 45, wherein the 2′-substituted sugar moietyof the modified nucleosides of the region of modified nucleosides is2′-MOE.

Embodiment 47

The compound of embodiment 43, wherein the modified nucleosides of theregion of modified nucleosides each comprise the same bicyclic sugarmoiety.

Embodiment 48

The compound of embodiment 47, wherein the bicyclic sugar moiety of themodified nucleosides of the region of modified nucleosides is selectedfrom LNA and cEt.

Embodiment 49

The compound of embodiment 41, wherein the modified nucleosides of theregion of modified nucleosides each comprises a sugar surrogate.

Embodiment 50

The compound of embodiment 49, wherein the sugar surrogate of themodified nucleosides of the region of modified nucleosides is amorpholino.

Embodiment 51

The compound of embodiment 50, wherein the sugar surrogate of themodified nucleosides of the region of modified nucleosides is a modifiedmorpholino.

Embodiment 52

The compound of any of embodiments 1 to 51, wherein the modifiednucleotide comprises no more than 4 contiguous naturally occurringnucleosides.

Embodiment 53

The compound of any of embodiments 1 to 52, wherein each nucleoside ofthe modified oligonucleotide is a modified nucleoside.

Embodiment 54

The compound of embodiment 53, wherein each modified nucleosidecomprises a modified sugar moiety.

Embodiment 55

The compound of embodiment 54, wherein the modified nucleosides of themodified oligonucleotide comprise the same modification as one another.

Embodiment 56

The compound of embodiment 55, wherein the modified nucleosides of themodified oligonucleotide each comprise the same 2′-substituted sugarmoiety.

Embodiment 57

The compound of embodiment 56, wherein the 2′-substituted sugar moietyof the modified oligonucleotide is selected from 2′-F, 2′-OMe, and2′-MOE.

Embodiment 58

The compound of embodiment 56, wherein the 2′-substituted sugar moietyof the modified oligonucleotide is 2′-MOE.

Embodiment 59

The compound of embodiment 55, wherein the modified nucleosides of themodified oligonucleotide each comprise the same bicyclic sugar moiety.

Embodiment 60

The compound of embodiment 59, wherein the bicyclic sugar moiety of themodified oligonucleotide is selected from LNA and cEt.

Embodiment 61

The compound of embodiment 55, wherein the modified nucleosides of themodified oligonucleotide each comprises a sugar surrogate.

Embodiment 62

The compound of embodiment 61, wherein the sugar surrogate of themodified oligonucleotide is a morpholino.

Embodiment 63

The compound of embodiment 61, wherein the sugar surrogate of themodified oligonucleotide is a modified morpholino.

Embodiment 64

The compound of any of embodiments 1 to 63, wherein the modifiedoligonucleotide comprises at least one modified internucleoside linkage.

Embodiment 65

The compound of embodiment 64, wherein each internucleoside linkage is amodified internucleoside linkage.

Embodiment 66

The compound of embodiment 64 or 65, comprising at least onephosphorothioate internucleoside linkage.

Embodiment 67

The compound of embodiment 64, wherein each internucleoside linkage is amodified internucleoside linkage and wherein each internucleosidelinkage comprises the same modification.

Embodiment 68

The compound of embodiment 67, wherein each internucleoside linkage is aphosphorothioate internucleoside linkage.

Embodiment 69

The compound of any of embodiments 1 to 68, comprising at least oneconjugate.

Embodiment 70

The compound of any of embodiments 1 to 69, consisting of the modifiedoligonucleotide.

Embodiment 71

The compound of any of embodiments 1 to 70, wherein the compoundmodulates splicing of the LRRK2 transcript.

Embodiment 72

The compound of any of embodiments 1 to 71, having a nucleobase sequencecomprising any of the sequences as set forth in SEQ ID NO:02, SEQ IDNO:08, or SEQ ID NO:09.

Embodiment 73

The compound of any of embodiments 1 to 72, having a nucleobase sequencecomprising any of the sequences as set forth in SEQ ID NO:01.

Embodiment 74

The compound of any of embodiments 1 to 72, having a nucleobase sequencecomprising any of the sequences as set forth in SEQ ID NO:06.

Embodiment 73

The compound of any of embodiment 73, having a nucleobase sequencecomprising SEQ ID NO:07.

Embodiment 74

A pharmaceutical composition comprising a compound according to any ofembodiments 1 to 73 and a pharmaceutically acceptable carrier ordiluent.

Embodiment 75

The pharmaceutical composition of embodiment 74, wherein thepharmaceutically acceptable carrier or diluent is sterile saline.

Embodiment 76

A method of modulating splicing of a LRRK2 transcript in a cellcomprising contacting the cell with a compound according to any ofembodiments 1 to 75.

Embodiment 77

The method of embodiment 76, wherein the cell is in vitro.

Embodiment 78

The method of embodiment 76, wherein the cell is in an animal.

Embodiment 79a

The method of any of embodiments 76 to 78, wherein the amount of LRRK2mRNA without exon 2 is increased.

Embodiment 79b

The method of any of embodiments 76 to 78, wherein the amount of LRRK2mRNA without exon 4 is increased.

Embodiment 80

The method of any of embodiments 76 to 78, wherein the amount of LRRK2mRNA without exon 31 is increased.

Embodiment 81

The method of any of embodiments 76 to 78, wherein the amount of LRRK2mRNA without exon 41 is increased.

Embodiment 82

The method of any of embodiments 76 to 81, wherein the LRRK2 transcriptis transcribed from a LRRK2 gene.

Embodiment 83

A method of modulating the expression of LRRK2 in a cell, comprisingcontacting the cell with a compound according to any of embodiments 1 to75.

Embodiment 84

The method of embodiment 83, wherein the cell is in vitro.

Embodiment 85

The method of embodiment 83, wherein the cell is in an animal.

Embodiment 86

A method comprising administering the compound according to any ofembodiments 1 to 73 or the pharmaceutical composition of embodiments 74or 75 to an animal.

Embodiment 87

The method of embodiment 86, wherein the administering step comprisesdelivering to the animal by intracerebroventricular injection,inhalation, parenteral injection or infusion, oral, subcutaneous orintramuscular injection, buccal, transdermal, transmucosal and topical.

Embodiment 88

The method of embodiment 87, wherein the administration is byintracerebroventricular injection.

Embodiment 89

The method of any of embodiments 86 to 88, wherein the animal has one ormore symptoms associated with Parkinson's disease.

Embodiment 90

The method of any of embodiments 86 to 88, wherein the administrationresults in amelioration of at least one symptom of Parkinson's disease.

Embodiment 91

The method of any of embodiments 86 to 90, wherein the animal is ahuman.

Embodiment 92

A method of treating Parkinson's disease, comprising administering thecompound according to any of embodiments 1 to 73 or the pharmaceuticalcomposition of embodiments 74 or 75 to an animal in need thereof.

Embodiment 93

Use of the compound according to any of embodiments 1 to 73 or thepharmaceutical composition of embodiments 74 or 75 for the preparationof a medicament for use in the treatment of Parkinson's disease.

Embodiment 94

Use of the compound according to any of embodiments 1 to 73 or thepharmaceutical composition of embodiments 74 or 75 for the preparationof a medicament for use in the amelioration of one or more symptomsassociated with Parkinson's disease.

Embodiment 95

A compound comprising a modified oligonucleotide consisting of 8 to 30linked nucleosides and having a nucleobase sequence comprising acomplementary region, wherein the complementary region comprises atleast 8 contiguous nucleobases complementary to an equal-length portionof a target region of a LRRK2 transcript.

Embodiment 96

The compound of embodiment 95, wherein the LRRK2 transcript comprisesthe nucleobase sequence of SEQ ID NO:03.

Embodiment 97

The compound of embodiment 95 or 96, wherein the complementary region ofthe modified oligonucleotide is 100% complementary to the target region.

Embodiment 98

The compound of any of embodiments 95 to 97, wherein the complementaryregion of the modified oligonucleotide comprises at least 10 contiguousnucleobases.

Embodiment 99

The compound of any of embodiments 95 to 97, wherein the complementaryregion of the modified oligonucleotide comprises at least 12 contiguousnucleobases.

Embodiment 100

The compound of any of embodiments 95 to 97, wherein the complementaryregion of the modified oligonucleotide comprises at least 14 contiguousnucleobases.

Embodiment 101

The compound of any of embodiments 95 to 97, wherein the complementaryregion of the modified oligonucleotide comprises at least 15 contiguousnucleobases.

Embodiment 102

The compound of any of embodiments 95 to 97, wherein the complementaryregion of the modified oligonucleotide comprises at least 16 contiguousnucleobases.

Embodiment 103

The compound of any of embodiments 95 to 97, wherein the complementaryregion of the modified oligonucleotide comprises at least 17 contiguousnucleobases.

Embodiment 104

The compound of any of embodiments 95 to 97, wherein the complementaryregion of the modified oligonucleotide comprises at least 18 contiguousnucleobases.

Embodiment 105

The compound of any of embodiments 95 to 104, wherein the nucleobasesequence of the modified oligonucleotide is at least 80% complementaryto an equal-length region of the LRRK2 transcript, as measured over theentire length of the oligonucleotide.

Embodiment 106

The compound of any of embodiments 95 to 104, wherein the nucleobasesequence of the modified oligonucleotide is at least 90% complementaryto an equal-length region of the LRRK2 transcript, as measured over theentire length of the oligonucleotide.

Embodiment 107

The compound of any of embodiments 95 to 104, wherein the nucleobasesequence of the modified oligonucleotide is 100% complementary to anequal-length region of the LRRK2 transcript, as measured over the entirelength of the oligonucleotide.

Embodiment 108

The compound of any of embodiments 95 to 107, wherein the target regionis within exon 2, exon 4, exon 31 or exon 41 of human LRRK2.

Embodiment 109

The compound of embodiment 108, wherein the target region is within exon31 of human LRRK2.

Embodiment 110

The compound of embodiment 108, wherein the target region is within exon41 of human LRRK2.

Embodiment 111

The compound of any of embodiments 95 to 107, wherein the modifiedoligonucleotide has a nucleobase sequence comprising any of thesequences as set forth in SEQ ID NO:02, SEQ ID NO:08, or SEQ ID NO:09.

Embodiment 112

The compound of any of embodiments 95 to 107, wherein the modifiedoligonucleotide has a nucleobase sequence consisting of the nucleobasesequence of any one of SEQ ID NO:01.

Embodiment 113

The compound of any of embodiments 95 to 107, wherein the modifiedoligonucleotide has a nucleobase sequence comprising the nucleobasesequence of SEQ ID NO:06.

Embodiment 114

The compound of any of embodiments 95 to 107, wherein the modifiedoligonucleotide has a nucleobase sequence consisting of the nucleobasesequence of SEQ ID NO:07.

Embodiment 115

The compound of any of embodiments 95 to 107, wherein the modifiedoligonucleotide has a nucleobase sequence comprising the nucleobasesequence of SEQ ID NO:01 SEQ ID NO:02, SEQ ID NO:06, SEQ ID NO:07, SEQID NO:08, or SEQ ID NO:09.

Embodiment 116

The compound of embodiment 115, wherein the modified oligonucleotide hasa nucleobase sequence consisting of the nucleobase sequence of SEQ IDNO:09.

Embodiment 117

The compound of any of embodiments 95 to 116, wherein the modifiedoligonucleotide comprises at least one modified nucleoside.

Embodiment 118

The compound of any of embodiments 95 to 117, wherein each nucleoside ofthe modified oligonucleotide is a modified nucleoside selected fromamong: 2′-OMe, 2′-F, and 2′-MOE or a sugar surrogate.

Embodiment 119

The compound of embodiment 118, wherein the modified nucleoside is2′-MOE.

Embodiment 120

The compound of embodiment 117, wherein the modified nucleoside is amorpholino.

Embodiment 121

The compound of embodiment 117, wherein at least one modified nucleosidecomprises a modified sugar moiety.

Embodiment 122

The compound of embodiment 121, wherein at least one modified sugarmoiety is a 2′-substituted sugar moiety.

Embodiment 123

The compound of embodiment 122, wherein the 2′-substitutent of at leastone 2′-substituted sugar moiety is selected from among: 2′-OMe, 2′-F,and 2′-MOE.

Embodiment 124

The compound of any of embodiments 122 to 123, wherein the2′-substituent of at least one 2′-substituted sugar moiety is a 2′-MOE.

Embodiment 125

The compound of any of embodiments 95 to 124, wherein at least onemodified sugar moiety is a bicyclic sugar moiety.

Embodiment 126

The compound of embodiment 125, wherein at least one bicyclic sugarmoiety is LNA or cEt.

Embodiment 127

The compound of any of embodiments 95 to 126, wherein at least one sugarmoiety is a sugar surrogate.

Embodiment 128

The compound of embodiment 127, wherein at least one sugar surrogate isa morpholino.

Embodiment 129

The compound of embodiment 128, wherein at least one sugar surrogate isa modified morpholino.

Embodiment 130

The compound of any of embodiments 95 to 129, wherein the modifiedoligonucleotide comprises at least 5 modified nucleosides, eachindependently comprising a modified sugar moiety.

Embodiment 131

The compound of any of embodiments 95 to 130, wherein the modifiedoligonucleotide comprises at least 10 modified nucleosides, eachindependently comprising a modified sugar moiety.

Embodiment 132

The compound of any of embodiments 95 to 130, wherein the modifiedoligonucleotide comprises at least 15 modified nucleosides, eachindependently comprising a modified sugar moiety.

Embodiment 133

The compound of any of embodiments 95 to 130, wherein each nucleoside ofthe modified oligonucleotide is a modified nucleoside, eachindependently comprising a modified sugar moiety.

Embodiment 134

The compound of any of embodiments 95 to 133, wherein the modifiedoligonucleotide comprises at least two modified nucleosides comprisingmodified sugar moieties that are the same as one another.

Embodiment 135

The compound of any of embodiments 95 to 133, wherein the modifiedoligonucleotide comprises at least two modified nucleosides comprisingmodified sugar moieties that are different from one another.

Embodiment 136

The compound of any of embodiments 95 to 136, wherein the modifiedoligonucleotide comprises a modified region of at least 5 contiguousmodified nucleosides.

Embodiment 137

The compound of any of embodiments 95 to 135, wherein the modifiedoligonucleotide comprises a modified region of at least 10 contiguousmodified nucleosides.

Embodiment 138

The compound of any of embodiments 95 to 135, wherein the modifiedoligonucleotide comprises a modified region of at least 15 contiguousmodified nucleosides.

Embodiment 139

The compound of any of embodiments 95 to 135, wherein the modifiedoligonucleotide comprises a modified region of at least 16 contiguousmodified nucleosides.

Embodiment 140

The compound of any of embodiments 95 to 135, wherein the modifiedoligonucleotide comprises a modified region of at least 17 contiguousmodified nucleosides.

Embodiment 141

The compound of any of embodiments 95 to 135, wherein the modifiedoligonucleotide comprises a modified region of at least 18 contiguousmodified nucleosides.

Embodiment 142

The compound of any of embodiments 95 to 135, wherein the modifiedoligonucleotide comprises a modified region of at least 20 contiguousmodified nucleosides.

Embodiment 143

The compound of any of embodiments 136 to 142, wherein each modifiednucleoside of the modified region has a modified sugar moietyindependently selected from among: 2′-F, 2′-OMe, 2′-MOE, cEt, LNA,morpholino, and modified morpholino.

Embodiment 144

The compound of any of embodiments 136 to 143, wherein the modifiednucleosides of the modified region each comprise the same modificationas one another.

Embodiment 145

The compound of embodiment 144, wherein the modified nucleosides of themodified region each comprise the same 2′-substituted sugar moiety.

Embodiment 146

The compound of embodiment 144, wherein the 2′-substituted sugar moietyof the modified nucleosides of the region of modified nucleosides isselected from 2′-F, 2′-OMe, and 2′-MOE.

Embodiment 147

The compound of embodiment 144, wherein the 2′-substituted sugar moietyof the modified nucleosides of the region of modified nucleosides is2′-MOE.

Embodiment 148

The compound of embodiment 144, wherein the modified nucleosides of theregion of modified nucleosides each comprise the same bicyclic sugarmoiety.

Embodiment 149

The compound of embodiment 148, wherein the bicyclic sugar moiety of themodified nucleosides of the region of modified nucleosides is selectedfrom LNA and cEt.

Embodiment 150

The compound of embodiment 144, wherein the modified nucleosides of theregion of modified nucleosides each comprises a sugar surrogate.

Embodiment 151

The compound of embodiment 150, wherein the sugar surrogate of themodified nucleosides of the region of modified nucleosides is amorpholino.

Embodiment 152

The compound of embodiment 150, wherein the sugar surrogate of themodified nucleosides of the region of modified nucleosides is a modifiedmorpholino.

Embodiment 153

The compound of any of embodiments 95 to 152, wherein the modifiednucleotide comprises no more than 4 contiguous naturally occurringnucleosides.

Embodiment 154

The compound of any of embodiments 95 to 152, wherein each nucleoside ofthe modified oligonucleotide is a modified nucleoside.

Embodiment 155

The compound of embodiment 154, wherein each modified nucleosidecomprises a modified sugar moiety.

Embodiment 156

The compound of embodiment 155, wherein the modified nucleosides of themodified oligonucleotide comprise the same modification as one another.

Embodiment 157

The compound of embodiment 156, wherein the modified nucleosides of themodified oligonucleotide each comprise the same 2′-substituted sugarmoiety.

Embodiment 158

The compound of embodiment 157, wherein the 2′-substituted sugar moietyof the modified oligonucleotide is selected from 2′-F, 2′-OMe, and2′-MOE.

Embodiment 159

The compound of embodiment 157, wherein the 2′-substituted sugar moietyof the modified oligonucleotide is 2′-MOE.

Embodiment 160

The compound of embodiment 158, wherein the modified nucleosides of themodified oligonucleotide each comprise the same bicyclic sugar moiety.

Embodiment 161

The compound of embodiment 160, wherein the bicyclic sugar moiety of themodified oligonucleotide is selected from LNA and cEt.

Embodiment 162

The compound of embodiment 156, wherein the modified nucleosides of themodified oligonucleotide each comprises a sugar surrogate.

Embodiment 163

The compound of embodiment 162, wherein the sugar surrogate of themodified oligonucleotide is a morpholino.

Embodiment 164

The compound of embodiment 162, wherein the sugar surrogate of themodified oligonucleotide is a modified morpholino.

Embodiment 165

The compound of any of embodiments 95 to 164, wherein the modifiedoligonucleotide comprises at least one modified internucleoside linkage.

Embodiment 166

The compound of embodiment 165, wherein each internucleoside linkage isa modified internucleoside linkage.

Embodiment 167

The compound of embodiment 165 or 166, comprising at least onephosphorothioate internucleoside linkage.

Embodiment 168

The compound of embodiment 166, wherein each internucleoside linkage isa modified internucleoside linkage and wherein each internucleosidelinkage comprises the same modification.

Embodiment 169

The compound of embodiment 168, wherein each internucleoside linkage isa phosphorothioate internucleoside linkage.

Embodiment 170

The compound of any of embodiments 95 to 169, comprising at least oneconjugate.

Embodiment 171

The compound of any of embodiments 95 to 170, consisting of the modifiedoligonucleotide.

Embodiment 172

The compound of any of embodiments 95 to 171, wherein the compoundmodulates splicing of the LRRK2 transcript.

Embodiment 173

A pharmaceutical composition comprising a compound according to any ofembodiments 95 to 172 and a pharmaceutically acceptable carrier ordiluent.

Embodiment 174

The pharmaceutical composition of embodiment 173, wherein thepharmaceutically acceptable carrier or diluent is sterile saline.

Embodiment 175

A method of modulating splicing of a LRRK2 transcript in a cellcomprising contacting the cell with a compound according to any ofembodiments 95 to 174.

Embodiment 176

The method of embodiment 175, wherein the cell is in vitro.

Embodiment 177

The method of embodiment 175, wherein the cell is in an animal.

Embodiment 178

The method of any of embodiments 175 to 177, wherein the amount of LRRK2mRNA without exon 2 is increased.

Embodiment 179

The method of any of embodiments 175 to 177, wherein the amount of LRRK2mRNA without exon 31 is increased.

Embodiment 180

The method of any of embodiments 175 to 177, wherein the amount of LRRK2mRNA with exon 41 is increased.

Embodiment 181

The method of any of embodiments 175 to 180, wherein the LRRK2transcript is transcribed from a LRRK2 gene.

Embodiment 182

A method of modulating the expression of LRRK2 in a cell, comprisingcontacting the cell with a compound according to any of embodiments 95to 174.

Embodiment 183

The method of embodiment 182, wherein the cell is in vitro.

Embodiment 184

The method of embodiment 182, wherein the cell is in an animal.

Embodiment 185

A method comprising administering the compound of any of embodiments 95to 172 to an animal.

Embodiment 186

The method of embodiment 185, wherein the administering step comprisesdelivering to the animal by intracerebroventricular injection,inhalation, parenteral injection or infusion, oral, subcutaneous orintramuscular injection, buccal, transdermal, transmucosal and topical.

Embodiment 187

The method of embodiment 185, wherein the administration isintracerebroventricular injection.

Embodiment 188

The method of any of embodiments 185 to 187, wherein the animal has oneor more symptoms associated with Parkinson's disease.

Embodiment 189

The method of any of embodiments 185 to 187, wherein the administrationresults in amelioration of at least one symptom of Parkinson's disease.

Embodiment 190

The method of any of embodiments 185 to 189, wherein the animal is ahuman.

Embodiment 191

A method of preventing or slowing one or more symptoms associated withParkinson's disease, comprising administering the compound according toany of embodiments 95 to 172 to an animal in need thereof.

Embodiment 192

The method of embodiment 191, wherein the animal is a human.

Embodiment 193

Use of the compound according to any of embodiments 95 to 172 for thepreparation of a medicament for use in the treatment of Parkinson'sdisease.

Embodiment 194

Use of the compound according to any of embodiments 95 to 172 for thepreparation of a medicament for use in the amelioration of one or moresymptoms associated with Parkinson's disease.

Embodiment 195

Use of the compound according to any of embodiments 95 to 172 for thepreparation of a medicament for use in the amelioration of one or moresymptoms associated with Parkinson's disease.

These and other features and advantages of the present disclosure willbe more fully understood from the following detailed description of theinvention taken together with the accompanying claims. It is noted thatthe scope of the claims is defined by the recitations therein and not bythe specific discussion of features and advantages set forth in thepresent description.

BRIEF DESCRIPTION OF THE DRAWINGS

A better understanding of the disclosure may be obtained in light of thefollowing drawings which are set forth for illustrative purposes, andshould not be construed as limiting the scope of the disclosure in anyway.

FIG. 1 is a schematic of the antisense oligonucleotides (ASOs) designedto block splicing to either exon 31 in patients with a LRRK2 R1441Cmutation or exon 41 in patients with a G2019S mutation. ASOs that blocksplicing of exon 41 will result in a frame-shift in the LRRK2 mRNA andprotein product, which will essentially eliminate LRRK2 expression. ASOsthat block splicing of exon 31 will eliminate the R1441C mutation andresult in the production of an alternative LRRK2 isoform predicted tohave lower kinase activity. Both of the ASO-induced LRRK2 mRNAtranscripts are predicted to mitigate disease symptoms by lessening thetoxic effects of the mutated LRRK2 protein.

FIG. 2 demonstrates antisense oligonucleotides, ASO-31-1 (SEQ ID NO: 01)and ASO-41-1 (SEQ ID NO: 02) successfully reduce full-length LRRK2expression by inducing skipping of LRRK2 exon 31 and 41 containing theR1441C and G2019S mutation, respectively, in fibroblast cells fromParkinson's patients.

FIG. 3A demonstrates that antisense oligonucleotides (ASO-41-1)successfully induces skipping of LRRK2 exon 41 containing the G2019Smutation in neurons derived from human induced pluripotent stem cells.

FIG. 3B demonstrates that antisense oligonucleotides (ASO-41-1)successfully induces skipping of LRRK2 exon 41 containing the G2019Smutation in neurons derived from human induced pluripotent stem cells.

FIG. 4A shows an electrophoretic analysis of the full length LRRK2 RNAproduct containing exon 41 (top lane) and LRRK2 RNA product with exon 41skipped (bottom lane) after ASO treatment. Treatment conditions: 411×-single treatment of cells with exon 41 ASO, 41 2×-double treatment ofcells with exon 41 ASO, 41-FL 1×-single treatment of cells with exon 41Fluorescent ASO, 41 2×-double treatment of cells with exon 41Fluorescent ASO, Ctl-FL 2×-double treatment of cells with non-targetFluorescent ASO, Ctl 2×-double treatment of cells with non-targetFluorescent ASO. Results demonstrate that LRRK2 exon 41 skipping inducedby antisense oligonucleotides (ASO) in iPS-derived neurons derived fromhealthy subject controls (CTRL 10A and 21.31) or PD patients carryingLRRK2 G2019S mutation (G2019S 29F and PD28).

FIG. 4B shows quantification of the percentage of exon 41 skipped whencompared to the amount of the full length LRRK2 RNA product pertreatment condition. Treatment conditions: 41 1×-single treatment ofcells with exon 41 ASO, 41 2×-double treatment of cells with exon 41ASO, 41-FL 1×-single treatment of cells with exon 41 Fluorescent ASO, 412×-double treatment of cells with exon 41 Fluorescent ASO, Ctl-FL2×-double treatment of cells with non-target Fluorescent ASO, Ctl2×-double treatment of cells with non-target Fluorescent ASO. Resultsdemonstrate that LRRK2 exon 41 skipping induced by antisenseoligonucleotides (ASO) in iPS-derived neurons derived from healthysubject controls (CTRL 10A and 21.31) or PD patients carrying LRRK2G2019S mutation (G2019S 29F and PD28).

FIG. 5A shows intracellular calcium levels were not altered in human iPSneurons carrying LRRK2 G2019S mutation compared to healthy subject (HS)controls. Detailed analysis showed no differences in the 1st and the 2ndcalcium peak amplitude upon KCl depolarization (arrows). Additionally,no difference was observed in calcium buffering upon a prolonged KCldepolarization indicating no difference in the binding andcompartmentalization of the unbound calcium. Results demonstrateiPS-derived neurons carrying LRRK2 G2019S mutation show altered calciumhomeostasis after ER calcium pump Serca inhibition, which can be rescuedby LRRK2 G2019S antisense oligonucleotide.

FIG. 5B shows intracellular calcium levels were not altered in human iPSneurons carrying LRRK2 G2019S mutation compared to healthy subject (HS)controls. Detailed analysis showed no differences in the 1st and the 2ndcalcium peak amplitude upon KCl depolarization (arrows). Additionally,no difference was observed in calcium buffering upon a prolonged KCldepolarization indicating no difference in the binding andcompartmentalization of the unbound calcium. Results demonstrateiPS-derived neurons carrying LRRK2 G2019S mutation show altered calciumhomeostasis after ER calcium pump Serca inhibition, which can be rescuedby LRRK2 G2019S antisense oligonucleotide.

FIG. 5C shows that upon Serca inhibition with 10 nM thapsigargin (THP),intracellular calcium levels were significantly increased in human iPSneurons carrying LRRK2 G2019S mutation compared to HS control. Detailedanalysis showed an increase in the 2nd calcium peak amplitude upon KCldepolarization and an increase in the unbound calcium levels indicatingdecreased calcium buffering. Results demonstrate iPS-derived neuronscarrying LRRK2 G2019S mutation show altered calcium homeostasis after ERcalcium pump Serca inhibition, which can be rescued by LRRK2 G2019Santisense oligonucleotide.

FIG. 5D shows that upon Serca inhibition with 10 nM thapsigargin (THP),intracellular calcium levels were significantly increased in human iPSneurons carrying LRRK2 G2019S mutation compared to HS control. Detailedanalysis showed an increase in the 2nd calcium peak amplitude upon KCldepolarization and an increase in the unbound calcium levels indicatingdecreased calcium buffering. Results demonstrate iPS-derived neuronscarrying LRRK2 G2019S mutation show altered calcium homeostasis after ERcalcium pump Serca inhibition, which can be rescued by LRRK2 G2019Santisense oligonucleotide.

FIG. 5E shows LRRK2 exon 41 G2019S targeting antisense oligonucleotide(G2019S ASO) normalizes the intracellular calcium levels in Sercablocked iPS-derived neurons carrying LRRK2 G2019S mutation. Both thecalcium amplitude during the 2nd KCl stimulation and the unbound calciumlevels show equal levels when compared to the HS control neurons treatedwith non-target ASO (NT ASO). Data was collected from fluoresceinpositive ASO transfected 4 PD patient iPS-derived neuronal linescarrying LRRK2 G2019S mutation and 3 healthy subject control lines; eachline represents a pool of 3 technical replicates per condition per line.Statistical analysis was performed using unpaired student T-test.*p<0.05.). Results demonstrate rescue of the LRRK2 G2019S inducedpathology in iPS-derived neurons using LRRK2 exon 41 skipping antisenseoligonucleotide strategy functional validation studies.

FIG. 5F shows LRRK2 exon 41 G2019S targeting antisense oligonucleotide(G2019S ASO) normalizes the intracellular calcium levels in Sercablocked iPS-derived neurons carrying LRRK2 G2019S mutation. Both thecalcium amplitude during the 2nd KCl stimulation and the unbound calciumlevels show equal levels when compared to the HS control neurons treatedwith non-target ASO (NT ASO). Data was collected from fluoresceinpositive ASO transfected 4 PD patient iPS-derived neuronal linescarrying LRRK2 G2019S mutation and 3 healthy subject control lines; eachline represents a pool of 3 technical replicates per condition per line.Statistical analysis was performed using unpaired student T-test.*p<0.05.). Results demonstrate rescue of the LRRK2 G2019S inducedpathology in iPS-derived neurons using LRRK2 exon 41 skipping antisenseoligonucleotide strategy functional validation studies.

FIG. 6A shows total ER-calcium levels were significantly decreased inthe iPS midbrain neurons carrying LRRK2 G2019S mutation compared to thehealthy subject control at baseline. Neurons were treated withnon-target negative control ASO (NT ASO). This decrease in the ERcalcium levels in the LRRK2 midbrain neurons was rescued with the G2019Sexon 41 antisense oligonucleotide (G2019S ASO). ER calcium levels weremeasured by total CEPIA-ER-GFP expression emission. Results demonstratethat iPS-derived midbrain neurons carrying LRRK2 G2019S mutation showdecreased total ER-calcium levels, which can be partially rescued byantisense oligonucleotide induced exon 41 skipping. Statistical analysiswas performed using unpaired student T-test. ****p<0.0001

FIG. 6B shows total ER-calcium levels were 20% significantly decreasedin the NT ASO treated iPS midbrain neurons carrying LRRK2 G2019Smutation compared to the healthy subject control after 24h of 10 nMthapsigargin induced Serca inhibition. Upon treatment with the G2019SASO, total ER-calcium levels in the LRRK2 midbrain neurons were 11%significantly lowered compared to the NT treated HS neurons suggesting apartial rescue of the ER calcium levels by the G2019S ASO. Data wascollected from one PD patient iPS-derived line carrying LRRK2 G2019Smutation and one healthy subject control line with the direct controlover the ASO treatment (NT vs G2019S); each neuronal genotype representsa pool of 3 technical replicates quantifying more than 100 neurons pereach condition. ER calcium levels were measured by total CEPIA-ER-GFPexpression emission. Results demonstrate that iPS-derived midbrainneurons carrying LRRK2 G2019S mutation show decreased total ER-calciumlevels, which can be partially rescued by antisense oligonucleotideinduced exon 41 skipping. Statistical analysis was performed usingunpaired student T-test. ****p<0.0001.

FIG. 6C shows ER-calcium levels in the control ASO (SEQ ID NO:05) andexon 41 ASO (SEQ ID NO:02) treated iPS neurons carrying LRRK2 G2019Smutation compared to the healthy subject (HS) control. ER calcium levelswere measured by total CEPIA-ER-GFP expression emission. Resultsdemonstrate that iPS-derived neurons carrying LRRK2 G2019S mutation showdecreased ER-calcium levels, which can be successfully rescued byantisense oligonucleotide induced exon 41 skipping. Statistical analysiswas performed using One way ANOVA with Holm-Sidak's multiple testingcorrection. *p<0.05.

FIG. 7A shows that neurite length is shortened in LRRK2 G2019S neuronsfollowing treatment with ER calcium pump Serca inhibitor thapsigargin(THP) by 24 hours and 48 hours post 10 nM and 100 nM treatment (N=3).One way Repeated measure ANOVA with Dunnett's multiple testingcorrection, *p<0.05, **p<0.01.

FIG. 7B shows that neurite length is unaffected in WT neurons (N=3)following treatment with a SERCA inhibitor, but rather, we observe anincrease of the neurite length in the vehicle treated cells. One wayRepeated measure ANOVA with Dunnett's multiple testing correction,**p<0.01.

FIG. 7C shows that neurite length is shortened in LRRK2 G2019S neuronsfollowing treatment with a SERCA inhibitor in an independent Parkinson'sdisease patient iPSC-derived neuronal cohort (N=4). One way Repeatedmeasure ANOVA with Dunnett's multiple testing correction, *p<0.05,**p<0.01.

FIG. 7D shows that neurite length is unaffected in gene correctedneurons following treatment with a SERCA inhibitor. One way Repeatedmeasure ANOVA with Dunnett's multiple testing correction.

FIG. 7E shows that neurite length is shortened in LRRK2 G2019S neuronsfollowing treatment with a non-specific control ASO following treatmentwith a SERCA inhibitor. One way Repeated measure ANOVA with Dunnett'smultiple testing correction. *p<0.05, **p<0.01.

FIG. 7F shows that exon 41 LRRK2 ASO induced skipping rescues neuriteoutgrowth THP induced collapse in LRRK2 G2019S neurons. One way Repeatedmeasure ANOVA with Dunnett's multiple testing correction.

FIG. 8A shows the DAF-FM fluorescence confluence and the nitric oxidelevels in LRRK2 G2019S iPS-derived neurons after valinomycin toxicity.Results demonstrate that an increase in RNS in LRRK2 G2019S neuronsafter mitochondrial depolarization induced through valinomycin toxicity.

FIG. 8B shows the DAF-FM fluorescence intensity and the nitric oxidelevels in LRRK2 G2019S iPS-derived neurons after valinomycin toxicity.Results demonstrate that an increase in RNS in LRRK2 G2019S neuronsafter mitochondrial depolarization induced through valinomycin toxicity.

FIG. 8C shows the nitric oxide levels in LRRK2 G2019S iPS-derivedneurons treated with exon 41 ASO (SEQ ID NO:02) and exon 31 ASO (SEQ IDNO:01). *p<0.05.

FIG. 9A shows the DAF-FM fluorescence intensity in T4.6 cell linesdemonstrating that nitric oxide levels are lower in LRRK2 G2019S genecorrected iPS neurons.

FIG. 9B shows the DAF-FM fluorescence intensity in T4.13 cell linesdemonstrating that nitric oxide levels are lower in LRRK2 G2019S genecorrected iPS neurons.

FIG. 9C shows the DAF-FM fluorescence intensity in IM1 cell linesdemonstrating that nitric oxide levels are lower in LRRK2 G2019S genecorrected iPS neurons.

FIG. 10A shows the MitoSOX™ fluorescence confluence and that super oxidelevels in LRRK2 G2019 iPS-derived neurons after valinomycin toxicity.Results demonstrate no difference in the ROS levels in LRRK2 G2019Sneurons compared to the healthy subject neurons after mitochondrialdepolarization induced through valinomycin toxicity.

FIG. 10B shows the MitoSOX™ fluorescence intensity and that super oxidelevels in LRRK2 G2019 iPS-derived neurons after valinomycin toxicity.Results demonstrate no difference in the ROS levels in LRRK2 G2019Sneurons compared to the healthy subject neurons after mitochondrialdepolarization induced through valinomycin toxicity.

FIG. 11A shows the MitoTracker® confluence in iPS control cells and thatdisruption of mitochondrial intracellular distribution in iPS-derivedneurons after mitochondrial depolarization.

FIG. 11B shows the MitoTracker® confluence in iPS LRRK2 G2019S cells andthat disruption of mitochondrial intracellular distribution iniPS-derived neurons after mitochondrial depolarization. Demonstratesaltered mitochondrial intracellular distribution in LRRK2 G2019SiPS-derived neurons after mitochondrial depolarization.

FIG. 12A shows the MitoTracker® distribution in iPS LRRK2 G2019S neuroncells and healthy control cells after 2 uM valinomycin toxicity.Demonstrates altered mitochondrial intracellular distribution in LRRK2G2019S iPS-derived neurons after mitochondrial depolarization induced byvalinomycin toxicity compared to healthy control neurons.

FIG. 12B shows the MitoTracker® distribution in iPS LRRK2 G2019S neuroncells and gene corrected neurons after 2 uM valinomycin toxicity.Demonstrates trend towards altered mitochondrial intracellulardistribution in LRRK2 G2019S iPS-derived neurons after mitochondrialdepolarization induced by valinomycin toxicity.

FIG. 13A shows the intracellular MitoTracker® distribution in iPS LRRK2G2019S neuron cells.

FIG. 13B shows the ERSE transcriptional activity in control and PD LRRK2G2019S cells and the decreased ER stress response in LRRK2 G2019Sneurons. 2-way ANOVA indicates genotype significance for allcomparisons. Results demonstrate that the ER stress response has loweractivation threshold in LRRK2 G2019S human neurons compared to healthysubject neurons after calcium store depletion induced by thapsigargin(THP).

FIG. 13C shows the ATF6 transcriptional activity in control and PD LRRK2G2019S cells and the decreased ER stress response in LRRK2 G2019Sneurons. 2-way ANOVA indicates genotype significance for allcomparisons. Results demonstrate that the ER stress response has loweractivation threshold in LRRK2 G2019S human neurons compared to healthysubject neurons after calcium store depletion induced by thapsigargin(THP).

FIG. 13D shows gene expression levels of UPR activators. 2-way ANOVAindicates genotype significance for all comparisons. Results demonstratethat the ER stress response has lower activation threshold in LRRK2G2019S human neurons compared to healthy subject neurons after calciumstore depletion induced by thapsigargin (THP).

FIG. 14A shows an image of Mitophagy Rosella bioprobe intracellularlocalization in the 293T Hek cells transfected with the LV-CMV-ATP3Rosella bioprobe. In addition to the red and green expression of theRosella bioprobe, cells were co-stained with Tom20 for mitochondrialdetection (Purple) and Hoechst (Blue) for nuclear identification.

FIG. 14B shows mitophagy measured with live cell imaging of the Rosellabioprobe in 293T HEK cells. Results demonstrate that the Rosellabioprobe can successfully indicate mitophagy in 293T cells, shown bylower levels of the Green/Red fluorescent ratio, after 1 μM rotenonetreatment of cells.

FIG. 14C shows mitophagy measured with Rosella bioprobe in healthysubject iPS-derived neurons. Results demonstrate that mitophagy can besuccessfully detected in healthy subject control human iPS neuronstreated with 1 μM rotenone using live cell imaging of Rosella bioprobe(indicated by lower levels of the Green/Red fluorescent ratio).

FIG. 15 is a schematic of the antisense oligonucleotides (ASOs) designedto block splicing to either exon 2 or exon 4 in patients carrying aLRRK2 R1441C and/or a LRRK2 G2019S mutation. ASOs that block splicing ofexon 2 or exon 4 will result in a frame-shift in the LRRK2 mRNA andprotein product, which will essentially eliminate LRRK2 expression. Bothof the ASO-induced LRRK2 mRNA transcripts are predicted to mitigatedisease symptoms by lessening the toxic effects of the mutated LRRK2protein.

FIG. 16 demonstrates that antisense oligonucleotides (ASOs: ex4, exon 4ASO, ATACACATATTACCTGAAGTTAGGA (SEQ ID NO:06); ex2, exon 2 ASO,AGTGAAAACAATGCCTTTACCTGCT (SEQ ID NO:07); and ex41-1, exon 41 ASO,AGACAGACCTGATCACCTACCTGGT (SEQ ID NO:02)) successfully induces skippingof LRRK2 exon 2, exon 4, and exon 41 containing either the R1441Cmutation or the G2019S mutation in fibroblast cell lines derived fromhuman patients carrying either the LRRK2 R1441C mutation or the LRRK2G2019S mutation.

FIG. 17A shows quantification of the percentage of exon 31, exon 4, andexon 2 skipped when compared to the amount of the full length LRRK2 RNAproduct per treatment condition. Fibroblast cells isolated from apatient having a LRRK2 R1441C mutation were treated with 7.5 μM, 22.5 μMor 45 μM of each ASO. ASO-5 (CTACCAGCCTACCATGTTACCTTGA; SEQ ID NO:01,targeting exon 31), ASO-23 (ATACACATATTACCTGAAGTTAGGA; SEQ ID NO:06,targeting exon 4), and ASO-45 (AGTGAAAACAATGCCTTTACCTGCT; SEQ ID NO:07,targeting exon 2).

FIG. 17B shows quantification of the percentage of exon 41, exon 4, andexon 2 skipped when compared to the amount of the full length LRRK2 RNAproduct per treatment condition. Fibroblast cells isolated from apatient having a LRRK2 G2019S mutation were treated with 7.5 μM, 22.5 μMor 45 μM of each ASO. ASO-6 (AGACAGACCTGATCACCTACCTGGT; SEQ ID NO:02,targeting exon 41), ASO-46 (GGTATCTGCCAGAAAATGCACAGGA; SEQ ID NO:08,targeting exon 41), ASO-23 (ATACACATATTACCTGAAGTTAGGA; SEQ ID NO:06,targeting exon 4), and ASO-45 (AGTGAAAACAATGCCTTTACCTGCT; SEQ ID NO:07,targeting exon 2).

FIG. 18 shows that ASOs directed to exon 41 induce dose responsive exon41 skipping in fibroblast cells isolated from a patient having a LRRK2G2019S mutation. Cells were treated with 7.5 μM, 15 μM, 22.5 μM, or 45μM of each ASO: ASO-6 (AGACAGACCTGATCACCTACCTGGT; SEQ ID NO:02,targeting exon 41), or ASO-46 (GGTATCTGCCAGAAAATGCACAGGA; SEQ ID NO:08,targeting exon 41).

FIG. 19 shows that an ASO directed to exon 31 induces exon skipping infibroblast cells isolated from a patient having a LRRK2 R1441C mutation.Cells were treated with 22.5 μM of ASO-31-1 (CTACCAGCCTACCATGTTACCTTGA;SEQ ID NO:01).

FIG. 20A shows quantification of the percentage of exon 31, exon 4, andexon 2 skipped when compared to the amount of the full length LRRK2 RNAproduct per treatment condition. Fibroblast cells isolated from apatient having a LRRK2 R1441C mutation were treated with 45 μM of eachASO: ASO-31-1 (CTACCAGCCTACCATGTTACCTTGA; SEQ ID NO:01), ASO-4-1(ATACACATATTACCTGAAGTTAGGA; SEQ ID NO:06), and ASO-2-1(AGTGAAAACAATGCCTTTACCTGCT; SEQ ID NO:07).

FIG. 20B shows quantification of the percentage of exon 41, exon 4, andexon 2 skipped when compared to the amount of the full length LRRK2 RNAproduct per treatment condition. Fibroblast cells isolated from apatient having a LRRK2 G2019S mutation were treated with 45 μM of eachASO: ASO-41-2 (GGTATCTGCCAGAAAATGCACAGGA; SEQ ID NO:08), ASO-4-1(ATACACATATTACCTGAAGTTAGGA; SEQ ID NO:06), and ASO-2-1(AGTGAAAACAATGCCTTTACCTGCT; SEQ ID NO:07).

FIG. 21 shows that an ASO specific to the LRRK2 G2019S mutation caninduce exon 41 skipping in fibroblast cells isolated from a patienthaving a LRRK2 G2019S mutation. Cells were treated with 7.5 μM of eachASO: ASO-284 (AATGCTGTAGTCAGCAATCTTTGCA; SEQ ID NO:09, targeting theLRRK2 G2019S mutation), ASO-6 (AGACAGACCTGATCACCTACCTGGT; SEQ ID NO:02,targeting exon 41).

FIG. 22A shows a concentration curve for an ASO specific to exon 41.ASO-6 (AGACAGACCTGATCACCTACCTGGT; SEQ ID NO:02).

FIG. 22B shows a concentration curve for an ASO specific to exon 41.ASO-46 (GGTATCTGCCAGAAAATGCACAGGA; SEQ ID NO:08).

FIG. 23A shows that an ASO (ASO 41-1) targeting exon 41 (at 1 μM dose)in PD patient fibroblasts having the LRRK2 G2019S mutation decreasesfibroblast cellular vulnerability to mitochondrial stress induced bymembrane depolarization (valinomycin toxicity). One way repeated ANOVAwith Dunnett's multiple testing correction; *p<0.05,

FIG. 23B shows quantification of the percentage of LRRK2 exon 41 skippedby ASO-41-1 in cells from healthy subjects (HS cells) and cells from PDpatients carrying the LRRK2 G2019S mutation (PD cells).

FIG. 24 shows that an ASO (ASO-6 (AGACAGACCTGATCACCTACCTGGT SEQ IDNO:02) directed to exon 41 induces dose responsive exon 41 skipping invarious human fibroblast cell lines. Cells were treated with 1 μM, 5 μM,or 15 μM of ASO-6. HS23 cells are healthy subject cells from donor 23;HS26 cells are healthy subject cells from donor 26; HS17 cells arehealthy subject cells from donor 17; HS30 cells are healthy subjectcells from donor 30; PD36 cells are Parkinson disease patient cells fromdonor 36 carrying the LRRK2 G2019S mutation; PD4 cells are Parkinsondisease patient cells from donor 4 carrying the LRRK2 G2019S mutation;PD09 cells are Parkinson disease patient cells from donor 09 carryingthe LRRK2 G2019S mutation; PD37 cells are Parkinson disease patientcells from donor 37 carrying the LRRK2 G2019S mutation.

FIG. 25 shows that LRRK2 exon 41 skipping increases the number of lysedmitochondria in human fibroblasts. Fibroblast cell lines isolated frompatients carrying a LRRK2 G2019S mutation (LRRK2 G2019S) and healthysubject controls (HS) cells were treated with 1 μM, 5 μM, or 15 μM ofASO-41-1.

FIG. 26A shows that an ASO (ASO-6) directed to exon 41 induces doseresponsive exon 41 skipping in various cell lines. Cells were treatedwith 1 μM, 5 μM, or 15 μM of ASO-6. HS23 cells are healthy subject cellsfrom donor 23; HS26 cells are healthy subject cells from donor 26; HS17cells are healthy subject cells from donor 17; PD36 cells are Parkinsondisease patient cells from donor 36 carrying the LRRK2 G2019S mutation;PD4 cells are Parkinson disease patient cells from donor 4 carrying theLRRK2 G2019S mutation; PD09 cells are Parkinson disease patient cellsfrom donor 09 carrying the LRRK2 G2019S mutation; PD37 cells areParkinson disease patient cells from donor 37 carrying the LRRK2 G2019Smutation.

FIG. 26B shows quantification of the percentage of LRRK2 exon 41 skippedinduced by ASO-6 in the HS cells and PD cells.

FIG. 27A shows mitochondrial acidification (an indication of rescue ofmitophagic flux) in PD patient derived fibroblasts carrying the LRRK2G2019S mutation upon exon 41 skipping induced by treatment with 1 μM ofASO#6.

FIG. 27B shows mitochondrial acidification (an indication of rescue ofmitophagic flux) in PD patient derived fibroblasts carrying the LRRK2G2019S mutation upon exon 41 skipping induced by treatment with 1 μM ofASO#6 imaged at 0.5 hours. One way ANOVA with Tukey's multiple testingcorrection; *p<0.05, **p<0.01, ***p<0.001.

FIG. 27C shows mitochondrial acidification (an indication of rescue ofmitophagic flux) in PD patient derived fibroblasts carrying the LRRK2G2019S mutation upon exon 41 skipping induced by treatment with 1 μM ofASO#6 imaged at 3 hours. One way ANOVA with Tukey's multiple testingcorrection; *p<0.05, ****p<0.0001.

FIG. 27D shows mitochondrial acidification (an indication of rescue ofmitophagic flux) in PD patient derived fibroblasts carrying the LRRK2G2019S mutation upon exon 41 skipping induced by treatment with 1 μM ofASO#6 imaged at 18 hours. One way ANOVA with Tukey's multiple testingcorrection; **p<0.01, ***p<0.001, ****p<0.0001.

FIG. 28A shows mitochondrial acidification (an indication of rescue ofmitophagic flux) in PD patient derived fibroblasts carrying the LRRK2G2019S mutation upon exon 2 skipping induced by treatment with 1 μM ofASO#45.

FIG. 28B shows mitochondrial acidification (an indication of rescue ofmitophagic flux) in PD patient derived fibroblasts carrying the LRRK2G2019S mutation upon exon 2 skipping induced by treatment with 1 μM ofASO#45 imaged at 0.5 hours. One way ANOVA with Tukey's multiple testingcorrection; **p<0.01.

FIG. 28C shows mitochondrial acidification (an indication of rescue ofmitophagic flux) in PD patient derived fibroblasts carrying the LRRK2G2019S mutation upon exon 2 skipping induced by treatment with 1 μM ofASO#45 imaged at 3 hours. One way ANOVA with Tukey's multiple testingcorrection; *p<0.05, **p<0.01, ****p<0.0001.

FIG. 28D shows mitochondrial acidification (an indication of rescue ofmitophagic flux) in PD patient derived fibroblasts carrying the LRRK2G2019S mutation upon exon 2 skipping induced by treatment with 1 μM ofASO#45 imaged at 18 hours. One way ANOVA with Tukey's multiple testingcorrection; *p<0.05, **p<0.01, ****p<0.0001.

FIG. 29A shows mitochondrial acidification (an indication of rescue ofmitophagic flux) in PD patient derived fibroblasts carrying the LRRK2R1441C mutation upon exon 31 skipping induced by treatment with 5 μM ofASO#5.

FIG. 29B shows mitochondrial acidification (an indication of rescue ofmitophagic flux) in PD patient derived fibroblasts carrying the LRRK2R1441C mutation upon exon 31 skipping induced by treatment with 5 μM ofASO#5 imaged at 0.5 hours. One way ANOVA with Tukey's multiple testingcorrection; **p<0.01.

FIG. 29C shows mitochondrial acidification (an indication of rescue ofmitophagic flux) in PD patient derived fibroblasts carrying the LRRK2R1441C mutation upon exon 31 skipping induced by treatment with 5 μM ofASO#5 imaged at 3 hours. One way ANOVA with Tukey's multiple testingcorrection; *p<0.05, **p<0.01.

FIG. 29D shows mitochondrial acidification (an indication of rescue ofmitophagic flux) in PD patient derived fibroblasts carrying the LRRK2R1441C mutation upon exon 31 skipping induced by treatment with 5 μM ofASO#5 imaged at 18 hours. One way ANOVA with Tukey's multiple testingcorrection; *p<0.05, **p<0.0, ***p<0.001.

FIG. 30A shows mitochondrial acidification (an indication of rescue ofmitophagic flux) in PD patient derived fibroblasts carrying the LRRK2R1441C mutation upon exon 2 skipping induced by treatment with 5 μM ofASO#45.

FIG. 30B shows mitochondrial acidification (an indication of rescue ofmitophagic flux) in PD patient derived fibroblasts carrying the LRRK2R1441C mutation upon exon 2 skipping induced by treatment with 5 μM ofASO#45 imaged at 0.5 hours. One way ANOVA with Tukey's multiple testingcorrection; *p<0.05.

FIG. 30C shows mitochondrial acidification (an indication of rescue ofmitophagic flux) in PD patient derived fibroblasts carrying the LRRK2R1441C mutation upon exon 2 skipping induced by treatment with 5 μM ofASO#45 imaged at 3 hours. One way ANOVA with Tukey's multiple testingcorrection; *p<0.05.

FIG. 31 shows a decrease in LRRK2 protein expression in PD patientderived fibroblasts carrying the LRRK2 G2019S mutation when treated withan ASO targeting exon 41. Treatment with 15 μM of ASO#6 results in adecrease in LRRK2 protein levels. Paired t-test, *p<0.05

FIG. 32A shows a decrease in LRRK2 protein expression in HS fibroblastsand PD patient derived fibroblasts carrying the LRRK2 G2019S mutationwhen treated with ASOs targeting exon 41 or exon 2. Treatment with 1 μMor 15 μM of ASO#6, or 15 μM of ASO#45 results in a decrease in LRRK2protein levels. One way ANOVA with Dunnett's multiple testingcorrection; *p<0.05.

FIG. 32B shows a decrease in LRRK2 protein expression in HS fibroblastsand PD patient derived fibroblasts carrying the LRRK2 G2019S mutationwhen treated with ASOs targeting exon 41 or exon 2. Treatment with 1 μMor 15 μM of ASO#6, or 15 μM of ASO#45 results in a decrease in LRRK2protein levels. One way ANOVA with Dunnett's multiple testingcorrection; *p<0.05.

FIG. 33 shows changes in phosphorylated LRRK2 protein levels(phosphorylation at Serine 935) in HS fibroblasts and PD patient derivedfibroblasts carrying the LRRK2 G2019S mutation when treated with ASOstargeting exon 41 or exon 2. Treatment with 1 μM or 15 of ASO#6, or 15μM of ASO#45 results in a change in phosphorylated LRRK2 protein levels.

FIG. 34A shows a decrease in LRRK2 protein expression in HS fibroblastsand PD patient derived fibroblasts carrying the LRRK2 R1441C mutationwhen treated with an ASO targeting exon 31. Treatment with 15 μM ofASO#5 results in a decrease in LRRK2 protein levels. Paired t-test,*p<0.05.

FIG. 34B shows a decrease in LRRK2 protein expression in PD patientderived fibroblasts carrying the LRRK2 R1441C mutation when treated withASOs targeting exon 31. Treatment with 15 μM of ASO#5 or 15 μM of ASO#45results in a decrease in LRRK2 protein levels.

FIG. 34C shows a decrease in LRRK2 protein expression in PD patientderived fibroblasts carrying the LRRK2 R1441C mutation when treated withan ASO targeting exon 31. Treatment with 15 μM of ASO#5 results in atrend of decrease in LRRK2 protein levels.

FIG. 34D shows changes in phosphorylated LRRK2 protein levels(phosphorylation at Serine 935) in HS cells and PD patient derivedfibroblasts carrying the LRRK2 R1441C mutation when treated with ASOstargeting exon 31 (treatment with 15 μM of ASO#5 or 15 of ASO#45).

FIG. 35 shows a decrease in LRRK2 protein expression in PD patientderived fibroblasts carrying the LRRK2 R1441C mutation when treated withASOs targeting exon 31 and exon 2. Treatment with 5 μM of ASO#5 orASO#45 results in a decrease in LRRK2 protein levels. One way ANOVA withDunnett's multiple testing correction & Paired T-test; *p<0.05.

FIG. 36 shows changes in phosphorylated LRRK2 protein levels(phosphorylation at Serine 935) in PD patient derived fibroblastscarrying the LRRK2 R1441C mutation when treated with ASOs targeting exon31 or exon 2 (treatment with 5 μM of ASO#5 or 5 μM of ASO#45).

FIG. 37 shows a decrease in LRRK2 protein expression in human HSfibroblasts treated with ASOs targeting exon 31 and exon 2. Treatmentwith 5 μM of ASO#5 or ASO#45 results in a decrease in LRRK2 proteinlevels. One way ANOVA with Tukey's multiple testing correction & PairedT-test; *p<0.05.

FIG. 38A shows that ASOs directed to exon 41 induce exon 41 skipping iniPS-derived neurons with WT LRRK2 or LRRK2 carrying the G2019S mutation.Cells were treated with 2×10 μM of an ASO specific to exon 41 (ASO#6;SEQ ID NO:02).

FIG. 38B shows a decrease in LRRK2 protein expression in iPS-derivedneurons from healthy subject and PD patient neurons carrying the LRRK2G2019S mutation treated with an ASO targeting exon 41 (Ex41) compared tonon-target ASO (Ctrl). Treatment with 2×10 μM of ASO#6 results in adecrease in LRRK2 protein levels. Ratio Paired t-test, *p<0.05.

FIG. 38C shows that ASOs directed to exon 41 induce exon 41 skipping iniPS-derived midbrain neurons with WT LRRK2 or LRRK2 carrying the G2019Smutation. Cells were treated with 2×10 μM of an ASO specific to exon 41(ASO#6; SEQ ID NO:02).

FIG. 39A shows that antisense oligonucleotides treatment of iPS-derivedneurons with an ASO targeting exon 31 successfully induces skipping ofLRRK2 exon 31. Cells were treated with 10 μM, 17.5 μM, or 25 μM of anASO specific to exon 31 (ASO#5; SEQ ID NO:01).

FIG. 39B shows quantification of the percentage of exon 31 skipped whentreated with an ASO targeting exon 31. Cells were treated with 10 μM,17.5 μM, or 25 μM of an ASO specific to exon 31 (ASO#5; SEQ ID NO:01).

FIG. 40 shows LRRK2 protein levels after exon 31 ASO induced skipping iniPS-derived neurons. ASO#5 decreases LRRK2 protein levels in iPS-derivedneurons expressing WT or R1441C mutant LRRK2.

Skilled artisans will appreciate that elements in the figures areillustrated for simplicity and clarity and have not necessarily beendrawn to scale. For example, the dimensions of some of the elements inthe figures can be exaggerated relative to other elements to helpimprove understanding of the embodiment(s) of the present disclosure.

DETAILED DESCRIPTION OF THE DISCLOSURE

The present disclosure relates to general compounds and methods to treatParkinson's disease in subjects using antisense oligonucleotides (ASOs)that induce specific pre-mRNA splicing events in LRRK2 gene transcriptsthat result in mRNAs that code for proteins that fully or partiallyrestore the function of LRRK2 (i.e., resulting in increased levels ofcorrectly localized LRRK2 protein at the plasma membrane and withincreased function). For example, some ASOs can base-pair with thetarget RNA and correct aberrant splicing caused by mutations, and otherASOs can induce skipping of exons with mutations that cause open readingframe-shifts. In such instances, skipping of the mutated exon using ASOscan restore the reading frame and generate an mRNA that codes for aLRRK2 isoform with partial function.

ASOs have been effectively used to alter pre-mRNA splicing (for review,Aartsma-Rus & van Ommen 2007; Smith et al., 2006). For example, ASOstargeted to cryptic splice sites created by mutations in the ATM genewere recently demonstrated to effectively redirect splicing to thecorrect splice site and improve protein expression (Du et al., 2007).The first clinical trials based on ASO-induced skipping of exons as atherapy for Duchenne muscular dystrophy (DMD) have shown success inincreasing dystrophin protein levels in muscle cells surrounding thesite of injection (van Deutekom et al., 2008). ASO-based therapies mayprovide a customizable approach to mutation-based treatments fordisease. The effectiveness of ASOs in modulating splicing in atherapeutically beneficial manner has been demonstrated for a number ofdiseases.

In an embodiment, this disclosure provides a therapeutic treatment ofhuman subjects having Parkinson's Disease by administering to the humansubject an ASO oligonucleotide having 8 to 30 linked nucleosides havinga nucleobase sequence comprising a complementary region comprising atleast 8 contiguous nucleobases complementary to a target region of equallength within an LRRK2 transcript. In an embodiment, the ASO may targetexon 2, exon 4, exon 31 or exon 41 of an LRRK2 transcript. Morespecifically, suitable ASOs will bind consecutive nucleotides of exon 31or exon 41 of LRRK2 via complementary base-pairing interactions and havea length of 8 to 30 nucleotides, more preferably 15 to 30 nucleotides,even more preferably 15 to 27 nucleotides and most preferably 15-25nucleotides or any range or combination of ranges therein. The ASOstargeting exon 2 or exon 4 induce exon 2 or exon 4 skipping,respectively, which will disrupt the LRRK2 reading frame and result in atruncated LRRK2 protein. Thus, exon 2 or exon 4 skipping induced by theASO results in an overall reduction in LRRK2 protein. The ASOssuccessfully reduce full-length LRRK2 expression by inducing skipping ofLRRK2 exon 31 and 41 containing LRRK2 with the R1441C or G2019Smutation, respectively.

The LRRK2 gene encodes a member of the leucine-rich repeat kinase familyand encodes a protein with an ankryin repeat region, a leucine-richrepeat (LRR) domain, a kinase domain, a DFG-like motif, a RAS domain, aGTPase domain, a MLK-like domain, and a WD40 domain. The protein ispresent largely in the cytoplasm but also associates with themitochondrial outer membrane. Mutations in LRRK2 are the most commonknown cause of familial and sporadic PD, accounting for approximately 5%of individuals with a family history of the disease and 3% of sporadiccases. It has been suggested that the G2019S mutation in LRRK2 resultsin stabilization of microtubules by tubulin-beta phosphorylation and mayrepresent a physiologic function of LRRK2 in neurons. Phosphorylation oftubulin was enhanced 3-fold by the LRRK2 G2019S mutation (i.e, a gain offunction mutation), which suggested that mutant LRRK2-inducedneurodegeneration in PD may be partly mediated by increasedphosphorylation of tubulin-beta, which may interfere with neuriteoutgrowth, axonal transport, and synapse formation.

Human (Homo sapiens) leucine-rich repeat kinase 2 (LRRK2) is located onchromosome 12 (genomic coordinates (GRCh38): 12:40,224,894-40,369,284).The gene is 9239 bp mRNA (RefSeq Gene ID: 120892; Official Symbol:LRRK2; Official Full Name: leucine rich repeat kinase 2) and is assignedNCBI Reference Sequence: NM_198578.3 (SEQ ID NO: 3); ACCESSION:NM_198578; Ensembl: ENSG00000188906. LRRK2 is also known as: PARKS;RIPK7; ROCO2; AURA17; DARDARIN; FLJ45829; DKFZp434H2111. Human LRRK2protein is assigned NCBI Reference Sequence: NP_940980.3 (2527 aa; SEQID NO: 4).

Antisense compounds, (e.g. antisense oligonucleotides (ASOs)) have beenused to modulate target nucleic acids. Antisense compounds comprising avariety of chemical modifications and motifs have been reported. Incertain instances, such compounds are useful as research tools,diagnostic reagents, and as therapeutic agents. In certain instances,antisense compounds have been shown to modulate protein expression bybinding to a target messenger RNA (mRNA) encoding the protein. Incertain instances, such binding of an antisense compound to its targetmRNA results in cleavage of the mRNA. Antisense compounds that modulateprocessing of a pre-mRNA have also been reported. Such antisensecompounds alter splicing, interfere with polyadenlyation or preventformation of the 5′-cap of a pre-mRNA.

Pre-mRNA splicing involves the precise and accurate removal of intronsfrom the pre-messenger RNA and the ligation of exons together afterintron removal to generate the mature mRNA which serves as the templatefor protein translation. Pre-mRNA splicing is a two-step reactioncarried out by a spliceosome complex comprising protein and small RNAcomponents which recognize conserved sequence elements within theintrons and exons of the RNA. Recognition of these sequence elements,including the 5′ splice site, 3′ splice site and branch point sequence,is the primary mechanism directing the correct removal of introns.

Splicing requires direct base-pairing between small nuclear RNA (snRNA)components of the spliceosome and the splice site nucleotides of themRNA. This interaction can be disrupted by gene mutations or byartificial blocking using short oligonucleotides complementary to theRNA. Such so called antisense oligonucleotides (ASOs), when designed tobe complementary to a splice sites, will compete for base-pairing withthe snRNAs, thereby blocking an essential step in splicing at the site.In this way, antisense oligonucleotides can potently block unwantedsplicing or redirect splicing to alternative splice sites, and canresult in mRNAs that code for proteins that fully or partially restorethe function to target transcripts. For example, antisenseoligonucleotides (ASOs) can be designed to block splicing to either exon31 in patients with a LRRK2 R1441C mutation or exon 41 in patients witha G2019S mutation. ASOs that block splicing of exon 41 will result in aframe-shift in the LRRK2 mRNA and protein product, which willessentially eliminate LRRK2 expression. ASOs that block splicing of exon31 will eliminate the R1441C mutation and result in the production of analternative LRRK2 isoform predicted to have lower kinase activity. Bothof the ASO-induced LRRK2 mRNA transcripts are predicted to mitigatedisease symptoms by lessening the toxic effects of the mutated LRRK2protein.

For example, ASOs can target the GLY2019SER (G2019S) mutation in exon 41of the LRRK2 gene. This mutation in LRRK2 is the most common known causeof familial and sporadic PD, accounting for approximately 5% ofindividuals with a family history of the disease and 3% of sporadiccases. The G2019S mutation lies within the mixed-lineage kinase-likedomain, and does not appear to alter the steady-state level, turnover,or intracellular localization of the LRRK2 protein, but the G2019Smutation appears to enhance protein kinase activity in a dominantnegative fashion. ASOs that block splicing of exon 41 will result in aframe-shift in the LRRK2 mRNA and protein product, which willessentially eliminate LRRK2 expression and mitigate PD symptoms bylessening the toxic effects of mutated LRRK2 protein. Non-limitingexamples of an ASO that prevents splicing of exon 41 are:

SEQ ID NO:02 (5′-AGACAGACCTGATCACCTACCTGGT-3′),

SEQ ID NO:08 (5′-GGTATCTGCCAGAAAATGCACAGGA-3′), or

SEQ ID NO:09 (5′-AATGCTGTAGTCAGCAATCTTTGCA-3′).

In another non-limiting example, ASOs can target the ILE2020THR (I2020T)mutation in exon 41 of the LRRK2 gene. The I2020T mutation in LRRK2 hasbeen found in PD patients. The I2020T mutation mutant protein showssignificantly increased (about 40%) autophosphorylation activitycompared to wildtype LRRK2, consistent with a gain of function. ASOsthat block splicing of exon 41 will result in a frame-shift in the LRRK2mRNA and protein product, which will essentially eliminate LRRK2expression and mitigate PD symptoms by lessening the toxic effects ofmutated LRRK2 protein.

In yet another non-limiting example, antisense oligonucleotides cantarget the ARG1441CYS (R1441C) mutation in exon 31 of the LRRK2 gene.This mutation has been observed in patients with Parkinson's disease.The R1441C mutation lies within the GTPase domain of LRRK2, and does notappear to alter the steady-state level, turnover, or intracellularlocalization of the LRRK2 protein, but the R1441C mutation appears toenhance protein kinase activity. ASOs that block splicing of exon 31will eliminate the R1441C mutation and result in the production of analternative LRRK2 isoform predicted to have lower kinase activity, whichshould mitigate disease symptoms by lessening the toxic effects of themutated LRRK2 protein. A non-limiting example of an ASO that preventssplicing of exon 31 is SEQ ID NO:01 (5′-CTACCAGCCTACCATGTTACCTTGA-3′).

In another non-limiting example, antisense oligonucleotides can targetthe ARG1441HIS (R1441H) mutation in exon 31 of the LRRK2 gene. TheR1441H mutation was found in PD patients. ASOs that block splicing ofexon 31 will eliminate the R1441H mutation and result in the productionof an alternative LRRK2 isoform predicted to have lower kinase activity,which should mitigate disease symptoms by lessening the toxic effects ofthe mutated LRRK2 protein.

In another non-limiting example, antisense oligonucleotides can targetthe ARG1441GLY (R1441G) mutation in exon 31 of the LRRK2 gene. TheR1441G mutation was found in 13.15% of 418 PD patients from the Basqueregion. ASOs that block splicing of exon 31 will eliminate the R1441Gmutation and result in the production of an alternative LRRK2 isoformpredicted to have lower kinase activity, which should mitigate diseasesymptoms by lessening the toxic effects of the mutated LRRK2 protein.

In yet another non-limiting example, the ASOs can target exon 2 or exon4, and induce exon 2 or exon 4 skipping, respectively, which willdisrupt the LRRK2 reading frame and result in a truncated LRRK2 protein.Thus, exon 2 or exon 4 skipping induced by the ASO results in an overallreduction in LRRK2 protein. A non-limiting example of an ASO targetingexon 2 is SEQ ID NO:07 (5′-AGTGAAAACAATGCCTTTACCTGCT-3′). A non-limitingexample of an ASO targeting exon 4 is SEQ ID NO:06(5′-ATACACATATTACCTGAAGTTAGGA-3′). Also see FIG. 15.

As used herein, “nucleoside” means a compound comprising a nucleobasemoiety and a sugar moiety. Nucleosides include, but are not limited to,naturally occurring nucleosides (as found in DNA and RNA) and modifiednucleosides. Nucleosides may be linked to a phosphate moiety.

As used herein, “antisense compound” or “antisense oligonucleotide(ASO)” means a compound comprising or consisting of an oligonucleotideat least a portion of which is complementary to a target nucleic acid towhich it is capable of hybridizing, resulting in at least one antisenseactivity.

As used herein, “antisense activity” means any detectable and/ormeasurable change attributable to the hybridization of an antisensecompound to its target nucleic acid. As used herein, “detecting” or“measuring” means that a test or assay for detecting or measuring isperformed. Such detection and/or measuring may result in a value ofzero. Thus, if a test for detection or measuring results in a finding ofno activity (activity of zero), the step of detecting or measuring theactivity has nevertheless been performed.

As used herein, “chemical modification” means a chemical difference in acompound when compared to a naturally occurring counterpart. Inreference to an oligonucleotide, chemical modification does not includedifferences only in nucleobase sequence. Chemical modifications ofoligonucleotides include nucleoside modifications (including sugarmoiety modifications and nucleobase modifications) and internucleosidelinkage modifications.

As used herein, “sugar moiety” means a naturally occurring sugar moietyor a modified sugar moiety of a nucleoside.

As used herein, “modified sugar moiety” means a substituted sugarmoiety, a bicyclic or tricyclic sugar moiety, or a sugar surrogate.

As used herein, “substituted sugar moiety” means a furanosyl comprisingat least one substituent group that differs from that of a naturallyoccurring sugar moiety. Substituted sugar moieties include, but are notlimited to, furanosyls comprising substituents at the 2′-position, the3′-position, the 5′-position and/or the 4′-position.

As used herein, “2′-substituted sugar moiety” means a furanosylcomprising a substituent at the 2′-position other than —H or —OH. Unlessotherwise indicated, a 2′-substituted sugar moiety is not a bicyclicsugar moiety (i.e., the 2′-substituent of a 2′-substituted sugar moietydoes not form a bridge to another atom of the furanosyl ring).

As used herein, “MOE” means —OCH₂CH₂OCH₃.

As used herein, “bicyclic sugar moiety” means a modified sugar moietycomprising a 4 to 7 membered ring (including but not limited to afuranosyl) comprising a bridge connecting two atoms of the 4 to 7membered ring to form a second ring, resulting in a bicyclic structure.In certain embodiments, the 4 to 7 membered ring is a sugar ring. Incertain embodiments, the 4 to 7 membered ring is a furanosyl. In certainsuch embodiments, the bridge connects the 2′-carbon and the 4′-carbon ofthe furanosyl.

As used herein, the term “sugar surrogate” means a structure that doesnot comprise a furanosyl and that is capable of replacing the naturallyoccurring sugar moiety of a nucleoside, such that the resultingnucleoside is capable of: (1) incorporation into an oligonucleotide and(2) hybridization to a complementary nucleoside. Such structures includerings comprising a different number of atoms than furanosyl (e.g., 4, 6,or 7-membered rings); replacement of the oxygen of a furanosyl with anon-oxygen atom (e.g., carbon, sulfur, or nitrogen); or both a change inthe number of atoms and a replacement of the oxygen. Such structures mayalso comprise substitutions corresponding to those described forsubstituted sugar moieties (e.g., 6-membered carbocyclic bicyclic sugarsurrogates optionally comprising additional substituents). Sugarsurrogates also include more complex sugar replacements (e.g., thenon-ring systems of peptide nucleic acid). Sugar surrogates includewithout limitation morpholino, modified morpholinos, cyclohexenyls andcyclohexitols.

As used here, the term “morpholino” means a sugar surrogate having thefollowing structure:

In certain embodiments, morpholinos may be modified, for example byadding or altering various substituent groups from the above morpholinostructure. Such sugar surrogates are referred to herein as “modifiedmorpholinos.” Morpholino comprising compositions are described in U.S.Pat. Nos. 5,142,047 and 5,185,444, incorporated by reference in theirentirety. In other embodiments, morpholinos may be unmodified. Forexample, the structure of an unmodified oligonucleotide is:

Combinations of modifications are also provided without limitation, suchas 2′-F-5′-methyl substituted nucleosides (see WO 2008/101157 for otherdisclosed 5′,2′-bis substituted nucleosides) and replacement of theribosyl ring oxygen atom with S and further substitution at the2′-position (see U.S. Patent Application US2005-0130923,) oralternatively 5′-substitution of a bicyclic nucleic acid (see WO2007/134181, wherein a 4′-CH.sub.2-O-2′ bicyclic nucleoside is furthersubstituted at the 5′ position with a 5′-methyl or a 5′-vinyl group).The synthesis and preparation of carbocyclic bicyclic nucleosides alongwith their oligomerization and biochemical studies have also beendescribed (see, e.g., Srivastava et al., J. Am. Chem. Soc. 2007,129(26), 8362-8379).

As used herein, “nucleotide” means a nucleoside further comprising aphosphate linking group.

As used herein, “linked nucleosides” may or may not be linked byphosphate linkages and thus includes, but is not limited to “linkednucleotides.” As used herein, “linked nucleosides” are nucleosides thatare connected in a continuous sequence (i.e. no additional nucleosidesare present between those that are linked).

As used herein, “nucleobase” means a group of atoms that can be linkedto a sugar moiety to create a nucleoside that is capable ofincorporation into an oligonucleotide, and wherein the group of atoms iscapable of bonding with a complementary naturally occurring nucleobaseof another oligonucleotide or nucleic acid. Nucleobases may be naturallyoccurring or may be modified.

As used herein, “heterocyclic base” or “heterocyclic nucleobase” means anucleobase comprising a heterocyclic structure.

As used herein, the terms, “unmodified nucleobase” or “naturallyoccurring nucleobase” means the naturally occurring heterocyclicnucleobases of RNA or DNA: the purine bases adenine (A) and guanine (G),and the pyrimidine bases thymine (T), cytosine (C) (including 5-methylC), and uracil (U).

As used herein, “modified nucleobase” means any nucleobase that is not anaturally occurring nucleobase.

As used herein, “modified nucleoside” means a nucleoside comprising atleast one chemical modification compared to naturally occurring RNA orDNA nucleosides. Modified nucleosides comprise a modified sugar moietyand/or a modified nucleobase.

As used herein, “bicyclic nucleoside” or “BNA” means a nucleosidecomprising a bicyclic sugar moiety.

As used herein, “constrained ethyl nucleoside” or “cEt” means anucleoside comprising a bicyclic sugar moiety comprising a4′-CH(CH₃)-0-2′bridge.

As used herein, “locked nucleic acid nucleoside” or “LNA” means anucleoside comprising a bicyclic sugar moiety comprising a4′-CH₂-0-2′bridge.

As used herein, “2′-substituted nucleoside” means a nucleosidecomprising a substituent at the 2′-position other than H or OH. Unlessotherwise indicated, a 2′-substituted nucleoside is not a bicyclicnucleoside.

As used herein, “2′-deoxynucleoside” means a nucleoside comprising 2′-Hfuranosyl sugar moiety, as found in naturally occurringdeoxyribonucleosides (DNA). In certain embodiments, a 2′-deoxynucleosidemay comprise a modified nucleobase or may comprise an RNA nucleobase(e.g., uracil).

As used herein, “oligonucleotide” means a compound comprising aplurality of linked nucleosides. In certain embodiments, anoligonucleotide comprises one or more unmodified ribonucleosides (RNA)and/or unmodified deoxyribonucleosides (DNA) and/or one or more modifiednucleosides.

As used herein “oligonucleoside” means an oligonucleotide in which noneof the internucleoside linkages contains a phosphorus atom. As usedherein, oligonucleotides include oligonucleosides.

As used herein, “modified oligonucleotide” means an oligonucleotidecomprising at least one modified nucleoside and/or at least one modifiedinternucleoside linkage.

As used herein “internucleoside linkage” means a covalent linkagebetween adjacent nucleosides in an oligonucleotide.

As used herein “naturally occurring internucleoside linkage” means a 3′to 5′ phosphodiester linkage.

As used herein, “modified internucleoside linkage” means anyinternucleoside linkage other than a naturally occurring internucleosidelinkage.

As used herein, “oligomeric compound” means a polymeric structurecomprising two or more substructures. In certain embodiments, anoligomeric compound comprises an oligonucleotide. In certainembodiments, an oligomeric compound comprises one or more conjugategroups and/or terminal groups. In certain embodiments, an oligomericcompound consists of an oligonucleotide.

As used herein, “terminal group” means one or more atom attached toeither, or both, the 3′ end or the 5′ end of an oligonucleotide. Incertain embodiments a terminal group is a conjugate group. In certainembodiments, a terminal group comprises one or more terminal groupnucleosides.

As used herein, “conjugate” means an atom or group of atoms bound to anoligonucleotide or oligomeric compound. In general, conjugate groupsmodify one or more properties of the compound to which they areattached, including, but not limited to, pharmacodynamic,pharmacokinetic, binding, absorption, cellular distribution, cellularuptake, charge and/or clearance properties.

As used herein, “conjugate linking group” means any atom or group ofatoms used to attach a conjugate to an oligonucleotide or oligomericcompound.

As used herein, “detectable and/or measurable activity” means astatistically significant activity that is not zero.

As used herein, “essentially unchanged” means little or no change in aparticular parameter, particularly relative to another parameter whichchanges much more. In certain embodiments, a parameter is essentiallyunchanged when it changes less than 5%. In certain embodiments, aparameter is essentially unchanged if it changes less than two-foldwhile another parameter changes at least ten-fold. For example, incertain embodiments, an antisense activity is a change in the amount ofa target nucleic acid. In certain such embodiments, the amount of anon-target nucleic acid is essentially unchanged if it changes much lessthan the target nucleic acid does, but the change need not be zero.

As used herein, “expression” means the process by which a geneultimately results in a protein. Expression includes, but is not limitedto, transcription, post-transcriptional modification (e.g., splicing,polyadenlyation, addition of 5′-cap), and translation.

As used herein, “target nucleic acid” means a nucleic acid molecule towhich an antisense compound hybridizes.

As used herein, “mRNA” means an RNA molecule that encodes a protein.

As used herein, “pre-mRNA” means an RNA transcript that has not beenfully processed into mRNA. Pre-RNA includes one or more intron.

As used herein, “transcript” means an RNA molecule transcribed from DNA.Transcripts include, but are not limited to mRNA, pre-mRNA, andpartially processed RNA.

As used herein, “targeting” or “targeted to” means the association of anantisense compound to a particular target nucleic acid molecule or aparticular region of a target nucleic acid molecule. An antisensecompound targets a target nucleic acid if it is sufficientlycomplementary to the target nucleic acid to allow hybridization underphysiological conditions.

As used herein, “nucleobase complementarity” or “complementarity” whenin reference to nucleobases means a nucleobase that is capable of basepairing with another nucleobase. For example, in DNA, adenine (A) iscomplementary to thymine (T). For example, in RNA, adenine (A) iscomplementary to uracil (U). In certain embodiments, complementarynucleobase means a nucleobase of an antisense compound that is capableof base pairing with a nucleobase of its target nucleic acid. Forexample, if a nucleobase at a certain position of an antisense compoundis capable of hydrogen bonding with a nucleobase at a certain positionof a target nucleic acid, then the position of hydrogen bonding betweenthe oligonucleotide and the target nucleic acid is considered to becomplementary at that nucleobase pair. Nucleobases comprising certainmodifications may maintain the ability to pair with a counterpartnucleobase and thus, are still capable of nucleobase complementarity.

As used herein, “non-complementary” in reference to nucleobases means apair of nucleobases that do not form hydrogen bonds with one another.

As used herein, “complementary” in reference to oligomeric compounds(e.g., linked nucleosides, oligonucleotides, or nucleic acids) means thecapacity of such oligomeric compounds or regions thereof to hybridize toanother oligomeric compound or region thereof through nucleobasecomplementarity under stringent conditions. Complementary oligomericcompounds need not have nucleobase complementarity at each nucleoside.Rather, some mismatches are tolerated. In certain embodiments,complementary oligomeric compounds or regions are complementary at 70%of the nucleobases (70% complementary). In certain embodiments,complementary oligomeric compounds or regions are 80% complementary. Incertain embodiments, complementary oligomeric compounds or regions are90% complementary. In certain embodiments, complementary oligomericcompounds or regions are 95% complementary. In certain embodiments,complementary oligomeric compounds or regions are 100% complementary.

As used herein, “hybridization” means the pairing of complementaryoligomeric compounds (e.g., an antisense compound and its target nucleicacid). While not limited to a particular mechanism, the most commonmechanism of pairing involves hydrogen bonding, which may beWatson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, betweencomplementary nucleobases.

As used herein, “specifically hybridizes” means the ability of anoligomeric compound to hybridize to one nucleic acid site with greateraffinity than it hybridizes to another nucleic acid site. In certainembodiments, an antisense oligonucleotide specifically hybridizes tomore than one target site.

As used herein, “percent complementarity” means the percentage ofnucleobases of an oligomeric compound that are complementary to anequal-length portion of a target nucleic acid. Percent complementarityis calculated by dividing the number of nucleobases of the oligomericcompound that are complementary to nucleobases at correspondingpositions in the target nucleic acid by the total length of theoligomeric compound.

As used herein, “percent identity” means the number of nucleobases in afirst nucleic acid that are the same type (independent of chemicalmodification) as nucleobases at corresponding positions in a secondnucleic acid, divided by the total number of nucleobases in the firstnucleic acid.

As used herein, “modulation” means a change of amount or quality of amolecule, function, or activity when compared to the amount or qualityof a molecule, function, or activity prior to modulation. For example,modulation includes the change, either an increase (stimulation orinduction) or a decrease (inhibition or reduction) in gene expression.As a further example, modulation of expression can include a change insplice site selection of pre-mRNA processing, resulting in a change inthe absolute or relative amount of a particular splice-variant comparedto the amount in the absence of modulation.

As used herein, “motif” means a pattern of chemical modifications in anoligomeric compound or a region thereof. Motifs may be defined bymodifications at certain nucleosides and/or at certain linking groups ofan oligomeric compound.

As used herein, “nucleoside motif” means a pattern of nucleosidemodifications in an oligomeric compound or a region thereof. Thelinkages of such an oligomeric compound may be modified or unmodified.Unless otherwise indicated, motifs herein describing only nucleosidesare intended to be nucleoside motifs. Thus, in such instances, thelinkages are not limited.

As used herein, “sugar motif” means a pattern of sugar modifications inan oligomeric compound or a region thereof.

As used herein, “linkage motif” means a pattern of linkage modificationsin an oligomeric compound or region thereof. The nucleosides of such anoligomeric compound may be modified or unmodified. Unless otherwiseindicated, motifs herein describing only linkages are intended to belinkage motifs. Thus, in such instances, the nucleosides are notlimited.

As used herein, “nucleobase modification motif” means a pattern ofmodifications to nucleobases along an oligonucleotide. Unless otherwiseindicated, a nucleobase modification motif is independent of thenucleobase sequence.

As used herein, “sequence motif” means a pattern of nucleobases arrangedalong an oligonucleotide or portion thereof. Unless otherwise indicated,a sequence motif is independent of chemical modifications and thus mayhave any combination of chemical modifications, including no chemicalmodifications.

As used herein, “type of modification” in reference to a nucleoside or anucleoside of a “type” means the chemical modification of a nucleosideand includes modified and unmodified nucleosides. Accordingly, unlessotherwise indicated, a “nucleoside having a modification of a firsttype” may be an unmodified nucleoside.

As used herein, “differently modified” mean chemical modifications orchemical substituents that are different from one another, includingabsence of modifications. Thus, for example, a MOE nucleoside and anunmodified DNA nucleoside are “differently modified,” even though theDNA nucleoside is unmodified. Likewise, DNA and RNA are “differentlymodified,” even though both are naturally-occurring unmodifiednucleosides. Nucleosides that are the same but for comprising differentnucleobases are not differently modified. For example, a nucleosidecomprising a 2′-OMe modified sugar and an unmodified adenine nucleobaseand a nucleoside comprising a 2′-OMe modified sugar and an unmodifiedthymine nucleobase are not differently modified.

As used herein, “the same type of modifications” refers to modificationsthat are the same as one another, including absence of modifications.Thus, for example, two unmodified DNA nucleoside have “the same type ofmodification,” even though the DNA nucleoside is unmodified. Suchnucleosides having the same type modification may comprise differentnucleobases.

As used herein, “pharmaceutically acceptable carrier or diluent” meansany substance suitable for use in administering to an animal. In certainembodiments, a pharmaceutically acceptable carrier or diluent is sterilesaline. In certain embodiments, such sterile saline is pharmaceuticalgrade saline.

Certain Motifs

In certain embodiments, the present invention provides oligomericcompounds comprising oligonucleotides. In certain embodiments, sucholigonucleotides comprise one or more chemical modification. In certainembodiments, chemically modified oligonucleotides comprise one or moremodified nucleosides. In certain embodiments, chemically modifiedoligonucleotides comprise one or more modified nucleosides comprisingmodified sugars. In certain embodiments, chemically modifiedoligonucleotides comprise one or more modified nucleosides comprisingone or more modified nucleobases. In certain embodiments, chemicallymodified oligonucleotides comprise one or more modified internucleosidelinkages. In certain embodiments, the chemically modifications (sugarmodifications, nucleobase modifications, and/or linkage modifications)define a pattern or motif. In certain embodiments, the patterns ofchemical modifications of sugar moieties, internucleoside linkages, andnucleobases are each independent of one another. Thus, anoligonucleotide may be described by its sugar modification motif,internucleoside linkage motif and/or nucleobase modification motif (asused herein, nucleobase modification motif describes the chemicalmodifications to the nucleobases independent of the sequence ofnucleobases).

Certain Sugar Motifs

In certain embodiments, oligonucleotides comprise one or more type ofmodified sugar moieties and/or naturally occurring sugar moietiesarranged along an oligonucleotide or region thereof in a defined patternor sugar modification motif. Such motifs may include any of the sugarmodifications discussed herein and/or other known sugar modifications.

In certain embodiments, the oligonucleotides comprise or consist of aregion having a gapmer sugar modification motif, which comprises twoexternal regions or “wings” and an internal region or “gap.” The threeregions of a gapmer motif (the 5′-wing, the gap, and the 3′-wing) form acontiguous sequence of nucleosides wherein at least some of the sugarmoieties of the nucleosides of each of the wings differ from at leastsome of the sugar moieties of the nucleosides of the gap. Specifically,at least the sugar moieties of the nucleosides of each wing that areclosest to the gap (the 3′-most nucleoside of the 5′-wing and the5′-most nucleoside of the 3′-wing) differ from the sugar moiety of theneighboring gap nucleosides, thus defining the boundary between thewings and the gap. In certain embodiments, the sugar moieties within thegap are the same as one another. In certain embodiments, the gapincludes one or more nucleoside having a sugar moiety that differs fromthe sugar moiety of one or more other nucleosides of the gap. In certainembodiments, the sugar modification motifs of the two wings are the sameas one another (symmetric gapmer). In certain embodiments, the sugarmodification motifs of the 5′-wing differs from the sugar modificationmotif of the 3′-wing (asymmetric gapmer). In certain embodiments,oligonucleotides comprise 2′-MOE modified nucleosides in the wings and2′-F modified nucleosides in the gap.

In certain embodiments, oligonucleotides are fully modified. In certainsuch embodiments, oligonucleotides are uniformly modified. In certainembodiments, oligonucleotides are uniform 2′-MOE. In certainembodiments, oligonucleotides are uniform 2′-F. In certain embodiments,oligonucleotides are uniform morpholino. In certain embodiments,oligonucleotides are uniform BNA. In certain embodiments,oligonucleotides are uniform LNA. In certain embodiments,oligonucleotides are uniform cEt.

In certain embodiments, oligonucleotides comprise a uniformly modifiedregion and additional nucleosides that are unmodified or differentlymodified. In certain embodiments, the uniformly modified region is atleast 5, 10, 15, 20 or 25 nucleosides in length. In certain embodiments,the uniform region is a 2′-MOE region. In certain embodiments, theuniform region is a 2′-F region. In certain embodiments, the uniformregion is a morpholino region. In certain embodiments, the uniformregion is a BNA region. In certain embodiments, the uniform region is aLNA region. In certain embodiments, the uniform region is a cEt region.

In certain embodiments, the oligonucleotide does not comprise more than4 contiguous unmodified 2′-deoxynucleosides. In certain circumstances,antisense oligonucleotides comprising more than 4 contiguous2′-deoxynucleosides activate RNase H, resulting in cleavage of thetarget RNA. In certain embodiments, such cleavage is avoided by nothaving more than 4 contiguous 2′-deoxynucleosides, for example, wherealteration of splicing and not cleavage of a target RNA is desired.

Certain Internucleoside Linkage Motifs

In certain embodiments, oligonucleotides comprise modifiedinternucleoside linkages arranged along the oligonucleotide or regionthereof in a defined pattern or modified internucleoside linkage motif.In certain embodiments, internucleoside linkages are arranged in agapped motif, as described above for sugar modification motif. In suchembodiments, the internucleoside linkages in each of two wing regionsare different from the internucleoside linkages in the gap region. Incertain embodiments the internucleoside linkages in the wings arephosphodiester and the internucleoside linkages in the gap arephosphorothioate. The sugar modification motif is independentlyselected, so such oligonucleotides having a gapped internucleosidelinkage motif may or may not have a gapped sugar modification motif andif it does have a gapped sugar motif, the wing and gap lengths may ormay not be the same.

In certain embodiments, oligonucleotides comprise a region having analternating internucleoside linkage motif. In certain embodiments,oligonucleotides of the present invention comprise a region of uniformlymodified internucleoside linkages. In certain such embodiments, theoligonucleotide comprises a region that is uniformly linked byphosphorothioate internucleoside linkages. In certain embodiments, theoligonucleotide is uniformly linked by phosphorothioate. In certainembodiments, each internucleoside linkage of the oligonucleotide isselected from phosphodiester and phosphorothioate. In certainembodiments, each internucleoside linkage of the oligonucleotide isselected from phosphodiester and phosphorothioate and at least oneinternucleoside linkage is phosphorothioate.

In certain embodiments, the oligonucleotide comprises at least 6phosphorothioate internucleoside linkages. In certain embodiments, theoligonucleotide comprises at least 8 phosphorothioate internucleosidelinkages. In certain embodiments, the oligonucleotide comprises at least10 phosphorothioate internucleoside linkages. In certain embodiments,the oligonucleotide comprises at least one block of at least 6consecutive phosphorothioate internucleoside linkages. In certainembodiments, the oligonucleotide comprises at least one block of atleast 8 consecutive phosphorothioate internucleoside linkages. Incertain embodiments, the oligonucleotide comprises at least one block ofat least 10 consecutive phosphorothioate internucleoside linkages. Incertain embodiments, the oligonucleotide comprises at least block of atleast one 12 consecutive phosphorothioate internucleoside linkages. Incertain such embodiments, at least one such block is located at the 3′end of the oligonucleotide. In certain such embodiments, at least onesuch block is located within 3 nucleosides of the 3′ end of theoligonucleotide.

Certain Nucleobase Modification Motifs

In certain embodiments, oligonucleotides comprise chemical modificationsto nucleobases arranged along the oligonucleotide or region thereof in adefined pattern or nucleobases modification motif. In certain suchembodiments, nucleobase modifications are arranged in a gapped motif. Incertain embodiments, nucleobase modifications are arranged in analternating motif. In certain embodiments, each nucleobase is modified.In certain embodiments, none of the nucleobases is chemically modified.

In certain embodiments, oligonucleotides comprise a block of modifiednucleobases. In certain such embodiments, the block is at the 3′-end ofthe oligonucleotide. In certain embodiments the block is within 3nucleotides of the 3′-end of the oligonucleotide. In certain suchembodiments, the block is at the 5′-end of the oligonucleotide. Incertain embodiments the block is within 3 nucleotides of the 5′-end ofthe oligonucleotide.

In certain embodiments, nucleobase modifications are a function of thenatural base at a particular position of an oligonucleotide. Forexample, in certain embodiments each purine or each pyrimidine in anoligonucleotide is modified. In certain embodiments, each adenine ismodified. In certain embodiments, each guanine is modified. In certainembodiments, each thymine is modified. In certain embodiments, eachcytosine is modified. In certain embodiments, each uracil is modified.

In certain embodiments, some, all, or none of the cytosine moieties inan oligonucleotide are 5-methyl cytosine moieties. Herein, 5-methylcytosine is not a “modified nucleobase.” Accordingly, unless otherwiseindicated, unmodified nucleobases include both cytosine residues havinga 5-methyl and those lacking a 5 methyl. In certain embodiments, themethylation state of all or some cytosine nucleobases is specified.

Certain Overall Lengths

In certain embodiments, the present invention provides oligomericcompounds including oligonucleotides of any of a variety of ranges oflengths. In certain embodiments, the invention provides oligomericcompounds or oligonucleotides consisting of X to Y linked nucleosides,where X represents the fewest number of nucleosides in the range and Yrepresents the largest number of nucleosides in the range. In certainsuch embodiments, X and Y are each independently selected from 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, and 50; provided that X<Y. For example, in certainembodiments, the invention provides oligomeric compounds which compriseoligonucleotides consisting of 8 to 9, 8 to 10, 8 to 11, 8 to 12, 8 to13, 8 to 14, 8 to 15, 8 to 16, 8 to 17, 8 to 18, 8 to 19, 8 to 20, 8 to21, 8 to 22, 8 to 23, 8 to 24, 8 to 25, 8 to 26, 8 to 27, 8 to 28, 8 to29, 8 to 30, 9 to 10, 9 to 11, 9 to 12, 9 to 13, 9 to 14, 9 to 15, 9 to16, 9 to 17, 9 to 18, 9 to 19, 9 to 20, 9 to 21, 9 to 22, 9 to 23, 9 to24, 9 to 25, 9 to 26, 9 to 27, 9 to 28, 9 to 29, 9 to 30, 10 to 11, 10to 12, 10 to 13, 10 to 14, 10 to 15, 10 to 16, 10 to 17, 10 to 18, 10 to19, 10 to 20, 10 to 21, 10 to 22, 10 to 23, 10 to 24, 10 to 25, 10 to26, 10 to 27, 10 to 28, 10 to 29, 10 to 30, 11 to 12, 11 to 13, 11 to14, 11 to 15, 11 to 16, 11 to 17, 11 to 18, 11 to 19, 11 to 20, 11 to21, 11 to 22, 11 to 23, 11 to 24, 11 to 25, 11 to 26, 11 to 27, 11 to28, 11 to 29, 11 to 30, 12 to 13, 12 to 14, 12 to 15, 12 to 16, 12 to17, 12 to 18, 12 to 19, 12 to 20, 12 to 21, 12 to 22, 12 to 23, 12 to24, 12 to 25, 12 to 26, 12 to 27, 12 to 28, 12 to 29, 12 to 30, 13 to14, 13 to 15, 13 to 16, 13 to 17, 13 to 18, 13 to 19, 13 to 20, 13 to21, 13 to 22, 13 to 23, 13 to 24, 13 to 25, 13 to 26, 13 to 27, 13 to28, 13 to 29, 13 to 30, 14 to 15, 14 to 16, 14 to 17, 14 to 18, 14 to19, 14 to 20, 14 to 21, 14 to 22, 14 to 23, 14 to 24, 14 to 25, 14 to26, 14 to 27, 14 to 28, 14 to 29, 14 to 30, 15 to 16, 15 to 17, 15 to18, 15 to 19, 15 to 20, 15 to 21, 15 to 22, 15 to 23, 15 to 24, 15 to25, 15 to 26, 15 to 27, 15 to 28, 15 to 29, 15 to 30, 16 to 17, 16 to18, 16 to 19, 16 to 20, 16 to 21, 16 to 22, 16 to 23, 16 to 24, 16 to25, 16 to 26, 16 to 27, 16 to 28, 16 to 29, 16 to 30, 17 to 18, 17 to19, 17 to 20, 17 to 21, 17 to 22, 17 to 23, 17 to 24, 17 to 25, 17 to26, 17 to 27, 17 to 28, 17 to 29, 17 to 30, 18 to 19, 18 to 20, 18 to21, 18 to 22, 18 to 23, 18 to 24, 18 to 25, 18 to 26, 18 to 27, 18 to28, 18 to 29, 18 to 30, 19 to 20, 19 to 21, 19 to 22, 19 to 23, 19 to24, 19 to 25, 19 to 26, 19 to 29, 19 to 28, 19 to 29, 19 to 30, 20 to21, 20 to 22, 20 to 23, 20 to 24, 20 to 25, 20 to 26, 20 to 27, 20 to28, 20 to 29, 20 to 30, 21 to 22, 21 to 23, 21 to 24, 21 to 25, 21 to26, 21 to 27, 21 to 28, 21 to 29, 21 to 30, 22 to 23, 22 to 24, 22 to25, 22 to 26, 22 to 27, 22 to 28, 22 to 29, 22 to 30, 23 to 24, 23 to25, 23 to 26, 23 to 27, 23 to 28, 23 to 29, 23 to 30, 24 to 25, 24 to26, 24 to 27, 24 to 28, 24 to 29, 24 to 30, 25 to 26, 25 to 27, 25 to28, 25 to 29, 25 to 30, 26 to 27, 26 to 28, 26 to 29, 26 to 30, 27 to28, 27 to 29, 27 to 30, 28 to 29, 28 to 30, or 29 to 30 linkednucleosides. In embodiments where the number of nucleosides of anoligomeric compound or oligonucleotide is limited, whether to a range orto a specific number, the oligomeric compound or oligonucleotide may,nonetheless further comprise additional other substituents. For example,an oligonucleotide comprising 8-30 nucleosides excludes oligonucleotideshaving 31 nucleosides, but, unless otherwise indicated, such anoligonucleotide may further comprise, for example one or moreconjugates, terminal groups, or other substituents. In certainembodiments, a gapmer oligonucleotide has any of the above lengths.

One of skill in the art will appreciate that certain lengths may not bepossible for certain motifs. For example: a gapmer having a 5′-wingregion consisting of four nucleotides, a gap consisting of at least sixnucleotides, and a 3′-wing region consisting of three nucleotides cannothave an overall length less than 13 nucleotides. Thus, one wouldunderstand that the lower length limit is 13 and that the limit of 10 in“10-20” has no effect in that embodiment. Further, where anoligonucleotide is described by an overall length range and by regionshaving specified lengths, and where the sum of specified lengths of theregions is less than the upper limit of the overall length range, theoligonucleotide may have additional nucleosides, beyond those of thespecified regions, provided that the total number of nucleosides doesnot exceed the upper limit of the overall length range. For example, anoligonucleotide consisting of 20-25 linked nucleosides comprising a5′-wing consisting of 5 linked nucleosides; a 3′-wing consisting of 5linked nucleosides and a central gap consisting of 10 linked nucleosides(5+5+10=20) may have up to 5 nucleosides that are not part of the5′-wing, the 3′-wing, or the gap (before reaching the overall lengthlimitation of 25). Such additional nucleosides may be 5′ of the 5′-wingand/or 3′ of the 3′ wing.

Certain Oligonucleotides

In certain embodiments, oligonucleotides of the present invention arecharacterized by their sugar motif, internucleoside linkage motif,nucleobase modification motif and overall length. In certainembodiments, such parameters are each independent of one another. Thus,each internucleoside linkage of an oligonucleotide having a gapmer sugarmotif may be modified or unmodified and may or may not follow the gapmermodification pattern of the sugar modifications. Thus, theinternucleoside linkages within the wing regions of a sugar-gapmer maybe the same or different from one another and may be the same ordifferent from the internucleoside linkages of the gap region. Likewise,such sugar-gapmer oligonucleotides may comprise one or more modifiednucleobase independent of the gapmer pattern of the sugar modifications.Herein if a description of an oligonucleotide or oligomeric compound issilent with respect to one or more parameter, such parameter is notlimited. Thus, an oligomeric compound described only as having a gapmersugar motif without further description may have any length,internucleoside linkage motif, and nucleobase modification motif. Unlessotherwise indicated, all chemical modifications are independent ofnucleobase sequence.

Certain Conjugate Groups

In certain embodiments, oligomeric compounds are modified by attachmentof one or more conjugate groups. In general, conjugate groups modify oneor more properties of the attached oligomeric compound including but notlimited to pharmacodynamics, pharmacokinetics, stability, binding,absorption, cellular distribution, cellular uptake, charge andclearance. Conjugate groups are routinely used in the chemical arts andare linked directly or via an optional conjugate linking moiety orconjugate linking group to a parent compound such as an oligomericcompound, such as an oligonucleotide. Conjugate groups includes withoutlimitation, intercalators, reporter molecules, polyamines, polyamides,polyethylene glycols, thioethers, polyethers, cholesterols,thiocholesterols, cholic acid moieties, folate, lipids, phospholipids,biotin, phenazine, phenanthridine, anthraquinone, adamantane, acridine,fluoresceins, rhodamines, coumarins and dyes. Certain conjugate groupshave been described previously, for example: cholesterol moiety(Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556),cholic acid (Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4,1053-1060), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al.,Ann. N.Y. Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med.Chem. Let., 1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al.,Nucl. Acids Res., 1992, 20, 533-538), an aliphatic chain, e.g.,do-decan-diol or undecyl residues (Saison-Behmoaras et al., EMBO J.,1991, 10, 1111-1118; Kabanov et al., FEBS Lett, 1990, 259, 327-330;Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethyl-ammonium1,2-di-0-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36:3651-3654; Shea et al., Nucl. Acids Res.,1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain(Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), oradamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995,36:3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta,1995, 1264, 229-237), or an octadecylamine orhexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277, 923-937).

In certain embodiments, a conjugate group comprises an active drugsubstance, for example, aspirin, warfarin, phenylbutazone, ibuprofen,suprofen, fen-bufen, ketoprofen, (S)-(+)-pranoprofen, carprofen,dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid, folinicacid, a benzothiadiazide, chlorothiazide, a diazepine, indo-methicin, abarbiturate, a cephalosporin, a sulfa drug, an antidiabetic, anantibacterial or an antibiotic.

In certain embodiments, conjugate groups are directly attached tooligonucleotides in oligomeric compounds. In certain embodiments,conjugate groups are attached to oligonucleotides by a conjugate linkinggroup. In certain such embodiments, conjugate linking groups, including,but not limited to, bifunctional linking moieties such as those known inthe art are amenable to the compounds provided herein. Conjugate linkinggroups are useful for attachment of conjugate groups, such as chemicalstabilizing groups, functional groups, reporter groups and other groupsto selective sites in a parent compound such as for example anoligomeric compound. In general a bifunctional linking moiety comprisesa hydrocarbyl moiety having two functional groups. One of the functionalgroups is selected to bind to a parent molecule or compound of interestand the other is selected to bind essentially any selected group such aschemical functional group or a conjugate group. In some embodiments, theconjugate linker comprises a chain structure or an oligomer of repeatingunits such as ethylene glycol or amino acid units. Examples offunctional groups that are routinely used in a bifunctional linkingmoiety include, but are not limited to, electrophiles for reacting withnucleophilic groups and nucleophiles for reacting with electrophilicgroups. In some embodiments, bifunctional linking moieties includeamino, hydroxyl, carboxylic acid, thiol, unsaturations (e.g., double ortriple bonds), and the like.

Some non-limiting examples of conjugate linking moieties includepyrrolidine, 8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) and6-aminohexanoic acid (AHEX or AHA). Other linking groups include, butare not limited to, substituted C₂-C₁₀ alkyl, substituted orunsubstituted C₂-C₁₀ alkenyl or substituted or unsubstituted C₂-C₁₀alkynyl, wherein a nonlimiting list of preferred substituent groupsincludes hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol,thioalkoxy, halogen, alkyl, aryl, alkenyl and alkynyl.

Conjugate groups may be attached to either or both ends of anoligonucleotide (terminal conjugate groups) and/or at any internalposition.

In certain embodiments, conjugate groups are at the 3′-end of anoligonucleotide of an oligomeric compound. In certain embodiments,conjugate groups are near the 3′-end. In certain embodiments, conjugatesare attached at the 3′end of an oligomeric compound, but before one ormore terminal group nucleosides. In certain embodiments, conjugategroups are placed within a terminal group.

In certain embodiments, the present invention provides oligomericcompounds. In certain embodiments, oligomeric compounds comprise anoligonucleotide. In certain embodiments, an oligomeric compoundcomprises an oligonucleotide and one or more conjugate and/or terminalgroups. Such conjugate and/or terminal groups may be added tooligonucleotides having any of the chemical motifs discussed above.Thus, for example, an oligomeric compound comprising an oligonucleotidehaving region of alternating nucleosides may comprise a terminal group.

Antisense Compounds

In certain embodiments, oligomeric compounds of the present inventionare antisense compounds. Such antisense compounds are capable ofhybridizing to a target nucleic acid, resulting in at least oneantisense activity. In certain embodiments, antisense compoundsspecifically hybridize to one or more target nucleic acid. In certainembodiments, a specifically hybridizing antisense compound has anucleobase sequence comprising a region having sufficientcomplementarity to a target nucleic acid to allow hybridization andresult in antisense activity and insufficient complementarity to anynon-target so as to avoid non-specific hybridization to any non-targetnucleic acid sequences under conditions in which specific hybridizationis desired (e.g., under physiological conditions for in vivo ortherapeutic uses, and under conditions in which assays are performed inthe case of in vitro assays).

In certain embodiments, the present invention provides antisensecompounds comprising oligonucleotides that are fully complementary tothe target nucleic acid over the entire length of the oligonucleotide.In certain embodiments, oligonucleotides are 99% complementary to thetarget nucleic acid. In certain embodiments, oligonucleotides are 95%complementary to the target nucleic acid. In certain embodiments, sucholigonucleotides are 90% complementary to the target nucleic acid. Incertain embodiments, such oligonucleotides are 85% complementary to thetarget nucleic acid. In certain embodiments, such oligonucleotides are80% complementary to the target nucleic acid. In certain embodiments, anantisense compound comprises a region that is fully complementary to atarget nucleic acid and is at least 80% complementary to the targetnucleic acid over the entire length of the oligonucleotide. In certainsuch embodiments, the region of full complementarity is from 6 to 14nucleobases in length.

In certain embodiments antisense compounds and antisenseoligonucleotides comprise single-strand compounds. In certainembodiments antisense compounds and antisense oligonucleotides comprisedouble-strand compounds.

Pharmaceutical Compositions

In certain embodiments, the present invention provides pharmaceuticalcompositions comprising one or more antisense compound. Thepharmaceutical composition may comprise a cocktail of antisensecompounds, wherein the cocktail comprises 2, 3, 4, 5, 6, 7, 8, 9, 10 ormore antisense compounds. In certain embodiments, such pharmaceuticalcomposition comprises a suitable pharmaceutically acceptable diluent orcarrier. In certain embodiments, a pharmaceutical composition comprisesa sterile saline solution and one or more antisense compound. In certainembodiments, such pharmaceutical composition consists of a sterilesaline solution and one or more antisense compound. In certainembodiments, the sterile saline is pharmaceutical grade saline. Incertain embodiments, a pharmaceutical composition comprises one or moreantisense compound and sterile water. In certain embodiments, apharmaceutical composition consists of one or more antisense compoundand sterile water. In certain embodiments, the sterile saline ispharmaceutical grade water. In certain embodiments, a pharmaceuticalcomposition comprises one or more antisense compound andphosphate-buffered saline (PBS). In certain embodiments, apharmaceutical composition consists of one or more antisense compoundand sterile phosphate-buffered saline (PBS). In certain embodiments, thesterile saline is pharmaceutical grade PBS.

In certain embodiments, antisense compounds may be admixed withpharmaceutically acceptable active and/or inert substances for thepreparation of pharmaceutical compositions or formulations.

Compositions and methods for the formulation of pharmaceuticalcompositions depend on a number of criteria, including, but not limitedto, route of administration, extent of disease, or dose to beadministered.

Pharmaceutical compositions comprising antisense compounds encompass anypharmaceutically acceptable salts, esters, or salts of such esters. Incertain embodiments, pharmaceutical compositions comprising antisensecompounds comprise one or more oligonucleotide which, uponadministration to an animal, including a human, is capable of providing(directly or indirectly) the biologically active metabolite or residuethereof. Accordingly, for example, the disclosure is also drawn topharmaceutically acceptable salts of antisense compounds, prodrugs,pharmaceutically acceptable salts of such prodrugs, and otherbioequivalents. Suitable pharmaceutically acceptable salts include, butare not limited to, sodium and potassium salts.

A prodrug can include the incorporation of additional nucleosides at oneor both ends of an oligomeric compound which are cleaved by endogenousnucleases within the body, to form the active antisense oligomericcompound.

Lipid moieties have been used in nucleic acid therapies in a variety ofmethods. In certain such methods, the nucleic acid is introduced intopreformed liposomes or lipoplexes made of mixtures of cationic lipidsand neutral lipids. In certain methods, DNA complexes with mono- orpoly-cationic lipids are formed without the presence of a neutral lipid.In certain embodiments, a lipid moiety is selected to increasedistribution of a pharmaceutical agent to a particular cell or tissue.In certain embodiments, a lipid moiety is selected to increasedistribution of a pharmaceutical agent to fat tissue. In certainembodiments, a lipid moiety is selected to increase distribution of apharmaceutical agent to muscle tissue.

In certain embodiments, pharmaceutical compositions provided hereincomprise one or more modified oligonucleotides and one or moreexcipients. In certain such embodiments, excipients are selected fromwater, salt solutions, alcohol, polyethylene glycols, gelatin, lactose,amylase, magnesium stearate, talc, silicic acid, viscous paraffin,hydroxymethylcellulose and polyvinylpyrrolidone.

In certain embodiments, a pharmaceutical composition provided hereincomprises a delivery system. Examples of delivery systems include, butare not limited to, liposomes and emulsions. Certain delivery systemsare useful for preparing certain pharmaceutical compositions includingthose comprising hydrophobic compounds. In certain embodiments, certainorganic solvents such as dimethylsulfoxide (DMSO) are used.

In certain embodiments, a pharmaceutical composition provided hereincomprises one or more tissue-specific delivery molecules designed todeliver the one or more pharmaceutical agents of the present inventionto specific tissues or cell types. For example, in certain embodiments,pharmaceutical compositions include liposomes coated with atissue-specific antibody.

In certain embodiments, a pharmaceutical composition provided hereincomprises a co-solvent system. Certain of such co-solvent systemscomprise, for example, benzyl alcohol, a nonpolar surfactant, awater-miscible organic polymer, and an aqueous phase. In certainembodiments, such co-solvent systems are used for hydrophobic compounds.A non-limiting example of such a co-solvent system is the VPD co-solventsystem, which is a solution of absolute ethanol comprising 3% w/v benzylalcohol, 8% w/v of the nonpolar surfactant Polysorbate 80™ and 65% w/vpolyethylene glycol 300. The proportions of such co-solvent systems maybe varied considerably without significantly altering their solubilityand toxicity characteristics. Furthermore, the identity of co-solventcomponents may be varied: for example, other surfactants may be usedinstead of Polysorbate 80™; the fraction size of polyethylene glycol maybe varied; other biocompatible polymers may replace polyethylene glycol,e.g., polyvinyl pyrrolidone; and other sugars or polysaccharides maysubstitute for dextrose.

In certain embodiments, a pharmaceutical composition provided herein isprepared for oral administration. In certain embodiments, pharmaceuticalcompositions are prepared for buccal administration.

In certain embodiments, a pharmaceutical composition is prepared foradministration by injection (e.g., intravenous, subcutaneous,intramuscular, etc.). In certain of such embodiments, a pharmaceuticalcomposition comprises a carrier and is formulated in aqueous solution,such as water or physiologically compatible buffers such as Hanks'ssolution, Ringer's solution, or physiological saline buffer. In certainembodiments, other ingredients are included (e.g., ingredients that aidin solubility or serve as preservatives). In certain embodiments,injectable suspensions are prepared using appropriate liquid carriers,suspending agents and the like. Certain pharmaceutical compositions forinjection are presented in unit dosage form, e.g., in ampoules or inmulti-dose containers. Certain pharmaceutical compositions for injectionare suspensions, solutions or emulsions in oily or aqueous vehicles, andmay contain formulatory agents such as suspending, stabilizing and/ordispersing agents. Certain solvents suitable for use in pharmaceuticalcompositions for injection include, but are not limited to, lipophilicsolvents and fatty oils, such as sesame oil, synthetic fatty acidesters, such as ethyl oleate or triglycerides, and liposomes. Aqueousinjection suspensions may contain substances that increase the viscosityof the suspension, such as sodium carboxymethyl cellulose, sorbitol, ordextran. Optionally, such suspensions may also contain suitablestabilizers or agents that increase the solubility of the pharmaceuticalagents to allow for the preparation of highly concentrated solutions.

In certain embodiments, a pharmaceutical composition provided hereincomprises an oligonucleotide in a therapeutically effective amount. Incertain embodiments, the therapeutically effective amount is sufficientto prevent, alleviate or ameliorate symptoms of a disease or to prolongthe survival of the subject being treated. Determination of atherapeutically effective amount is well within the capability of thoseskilled in the art.

In certain embodiments, one or more modified oligonucleotide providedherein is formulated as a prodrug. In certain embodiments, upon in vivoadministration, a prodrug is chemically converted to the biologically,pharmaceutically or therapeutically more active form of anoligonucleotide. In certain embodiments, prodrugs are useful becausethey are easier to administer than the corresponding active form. Forexample, in certain instances, a prodrug may be more bioavailable (e.g.,through oral administration) than is the corresponding active form. Incertain instances, a prodrug may have improved solubility compared tothe corresponding active form. In certain embodiments, prodrugs are lesswater soluble than the corresponding active form. In certain instances,such prodrugs possess superior transmittal across cell membranes, wherewater solubility is detrimental to mobility. In certain embodiments, aprodrug is an ester. In certain such embodiments, the ester ismetabolically hydrolyzed to carboxylic acid upon administration. Incertain instances the carboxylic acid containing compound is thecorresponding active form. In certain embodiments, a prodrug comprises ashort peptide (polyaminoacid) bound to an acid group. In certain of suchembodiments, the peptide is cleaved upon administration to form thecorresponding active form.

In certain embodiments, the present invention provides compositions andmethods for reducing the amount or activity of a target nucleic acid ina cell. In certain embodiments, the cell is in an animal. In certainembodiments, the animal is a mammal. In certain embodiments, the animalis a rodent. In certain embodiments, the animal is a primate. In certainembodiments, the animal is a non-human primate. In certain embodiments,the animal is a human. In certain embodiments, the animal is a mouse.

In certain embodiments, the present invention provides methods ofadministering a pharmaceutical composition comprising an oligomericcompound of the present invention to an animal. Suitable administrationroutes include, but are not limited to, oral, rectal, transmucosal,transdermal, intestinal, enteral, topical, suppository, throughinhalation, intrathecal, intracerebroventricular, intraperitoneal,intranasal, intratumoral, and parenteral (e.g., intravenous,intramuscular, intramedullary, and subcutaneous). In certainembodiments, a pharmaceutical composition is prepared forintracerebroventricular administration or intracerebroventricularinjection. In such embodiments penetrants appropriate to the blood-brainbarrier to be permeated are used in the formulation. Such penetrants aregenerally known in the art. In certain embodiments, pharmaceuticalcompositions are administered to achieve local rather than systemicexposures.

In certain embodiments, a pharmaceutical composition is administered toan animal having at least one symptom associated with Parkinson'sdisease. In some embodiments, the animal has a mutation in the LRRK2gene and protein encoded by the mutated gene. Non-limiting examples ofsuch mutants include, but are not limited to, G2019S, I2020T, R1441C,R1441H, or R1141G. In other embodiments, the mutation can be anymutation in LRRK2 associated with Parkinson's disease. In certainembodiments, such administration results in amelioration of at least onesymptom. In certain embodiments, administration of a pharmaceuticalcomposition to an animal results in an increase in functional LRRK2protein in a cell. In certain embodiments, the administration of certainantisense oligonucleotides (ASOs) delays the onset of Parkinson'sdisease. In certain embodiments, the administration of certain antisenseoligonucleotides prevents the onset of Parkinson's disease. In certainembodiments, the administration of certain antisense oligonucleotidesrescues cellular phenotype.

While certain compounds, compositions and methods described herein havebeen described with specificity in accordance with certain embodiments,the following examples serve only to illustrate the compounds describedherein and are not intended to limit the same. Each of the references,GenBank accession numbers, and the like recited in the presentapplication is incorporated herein by reference in its entirety.Although the sequence listing accompanying this filing identifies eachsequence as either “RNA” or “DNA” as required, in reality, thosesequences may be modified with any combination of chemicalmodifications. One of skill in the art will readily appreciate that suchdesignation as “RNA” or “DNA” to describe modified oligonucleotides is,in certain instances, arbitrary. For example, an oligonucleotidecomprising a nucleoside comprising a 2′-OH sugar moiety and a thyminebase could be described as a DNA having a modified sugar (2′-OH for thenatural 2′-H of DNA) or as an RNA having a modified base (thymine(methylated uracil) for natural uracil of RNA).

Accordingly, nucleic acid sequences provided herein, including, but notlimited to those in the sequence listing, are intended to encompassnucleic acids containing any combination of natural or modified RNAand/or DNA, including, but not limited to such nucleic acids havingmodified nucleobases. By way of further example and without limitation,an oligomeric compound having the nucleobase sequence

EXAMPLES

The Examples that follow are illustrative of specific embodiments of theinvention, and various uses thereof. They are set forth for explanatorypurposes only, and are not to be taken as limiting the invention.

Methods

Antisense Oligonucleotides (ASOs).

ASOs were filtered, selectively precipitated, resuspended in pure water,and freeze dried to remove all contaminants. No acids or salts were usedin purification. ASOs with phosphorodiamidate morpholino (PMO)chemistries were generated by GeneTools LLC and were dissolved in 0.9%saline.

Cell Culture and Transfection.

Primary fibroblast cell lines established from non-disease (HealthySubject (HS) Control; CTRL) or a Parkinson's disease patient (G2019S orR1441C) skin biopsy were transfected with ASOs (1.0 μM, 5 μM, 7.5 μM, 15μM, 22.5 μM, or 45 μM final concentration) using Endo-Portertransfection reagent (GeneTools) according to manufacturer's protocol(see “Endo-Porter Delivery of Morpholino Oligos” from Gene Tools, LLC,14 Sep. 2012, pages 1-4). RNA was collected 48 hours post-transfection.IPS-derived neurons were treated with the LRRK2 G2019S exon 41anti-sense oligonucleotide with the sequence5′-AGACAGACCTGATCACCTACCTGGT-3′ (SEQ ID NO: 2) and a non-sense controloligonucleotide sequence 5′-CCTCTTACCTCAGTTACAATTTATA-3′ (SEQ ID NO: 5)either once (5 days post differentiation, DIV37) or two times (2 and 5days post end of the differentiation (DIV37 and DIV41)). Each timeneurons were treated with 10 μM LRRK2 ASOs using Endo-Porter deliverymethod according to the manufacturer's protocol (Gene Tools, 4 μl per 1mL solution). Exon skipping efficiency was also assessed by comparingbetween the non-tag and the carboxyflourescein-3′ tag ASO sequences. RNAwas collected 48 hours post the second transfection (DIV43).

RNA Isolation and Analysis.

RNA was isolated from cells using TRIZOL™ reagent (Life Technologies,Carlsbad, Calif.) according to the manufacturer's protocol, followed byreverse transcription with GoScript™ reverse transcription system(Promega, Madison, Wis.). Radiolabeled PCR was carried out using primersspecific for human LRRK2 region encompassing the ASO target exon. PCRproducts were separated by polyacrylamide gel electrophoresis and bandson gels were quantitated by densitometry analysis using Image J softwareor phosphorimage analysis using a TYPHOON™ phosphorimager.

Differentiation of Neurons from Human iPSCs.

Human induced pluripotent stem cells (iPS) cells derived from biopsiedfibroblasts from Parkinson's patients and healthy subject controls(Cooper et al., 2012, “Pharmacological Rescue of Mitochondrial Deficitsin iPSC-Derived Neural Cells from Patients with Familial Parkinson'sDisease,” Sci Transl Med. 2012, 4; 4(141):141ra90; see Table 1) weregrown on a feeder layer of mouse embryonic fibroblasts (MEFs)(GlobalStem, GSC-6001G) in HESCM media consisting of DMEM/F-12 (LifeTechnologies, 11330-032), 20% Knockout Serum (Life Technologies,10828-028), Penicillin-Streptomycin (Life Technologies, 15140-122), 1 mML-Glutamine (Life Technologies, 25030-081), 55 μM β-Mercaptoethanol(Gibco, 21985-023), and MEM non-essential amino acids (LifeTechnologies, 11140-050). LRRK2 G2019S gene corrected iPSC lines wereobtained from and previously characterized by Reinhardt et al., 2013,(“Genetic Correction of a LRRK2 Mutation in Human iPSCs LinksParkinsonian Neurodegeneration to ERK-Dependent Changes in GeneExpression.” Cell Stem Cell 12, 354-367). Table 1 provides an overviewof the iPS cell lines used.

TABLE 1 List of the human iPSC lines used in phenotypic assays Line NameGenotype 10A Control (CTRL) 21.31 Control (CTRL) 21.35 Control (CTRL) 9ALRRK2 G2019S 2F LRRK2 R1441C 3C LRRK2 R1441C PD28 LRRK2 G2019S PD28Nurr1:GFP LRRK2 G2019S 29F LRRK2 G2019S T4.6Mut LRRK2 G2019S T4.13MutLRRK2 G2019S IM1Mut LRRK2 G2019S IM2Mut LRRK2 G2019S T4.6.10GC LRRK2gene correction T4.6.43GC LRRK2 gene correction T4.13.10GC LRRK2 genecorrection IM1GC LRRK2 gene correction IM2GC LRRK2 gene correction

Embryoid Bodies (EB) Protocol for Generation of Neural Cells.

Neuronal cells were differentiated from induced pluripotent stem cells(iPSCs), following the procedures and modified from Brennand et al. 2011(“Modeling schizophrenia using human induced pluripotent stem cells,”Nature, 2011 May 12; 473(7346):221-5). In brief, iPSC colonies weredissociated using 1 mg/mL Collagenase IV (Life Technologies, 17104-019),resuspended in N2 GlutaMAX medium (DMEM GlutaMAX™, Life Technologies,10565-042; N2, Life Technologies, 17502-048) supplemented with 100 nMLDN (Stemgent, 04-0074-02), and plated on low attachment 6 well plates(Corning, 3471) to initiate suspension culture of embryoid bodies. OnDIV7 the embryoid bodies were replated for rosette formation onto 6 wellplates (Corning, 353046) coated with poly-L-ornithine (PLO, 15% in PBS,Sigma, P4957) and mouse laminin (1 μg/mL in DMEM/F-12, Sigma, L2020) inthe presence of N2 medium supplemented with 1 μg/mL laminin. On DIV 14,rosettes were cut manually and passaged onto new PLO-laminin coatedplates for expansion in DMEM/F12 media supplemented with N2, B-27 (LifeTechnologies, 17504-044), 2 μg/mL human recombinant bFGF (LifeTechnologies, 13256-029) and 1 μg/mL laminin. The final neuraldifferentiation phase was started by rosettes dissociation on DIV 21.Rosettes were dissociated using Accutase® (Millipore, SCR005) and platedonto PLO-laminin coated 6 well plates at a density of 300,000 cells perwell in neural differentiation media containing DMEM/F12, N2 and B27supplement, 1 mM dibutyry cyclic AMP (cAMP) (Enzo Life Sciences,BML-CN125-0100), 20 ng/mL BDNF (PreproTech, 450-02) and 200 μM Ascorbicacid (Sigma, A4034) (Neural Differentiation media). Cells were harvestedon DIV 35 by dissociation using Accutase® for phenotypic assays.

Generation of Ventral Midbrain Dopaminergic Neurons.

Midbrain dopaminergic neuron differentiation was initiated followingpreviously published protocols by Cooper et al., 2010 (“Differentiationof human ES and Parkinson's disease iPS cells into ventral midbraindopaminergic neurons requires a high activity form of SHH, FGF8a andspecific regionalization by retinoic acid,” Molecular and CellularNeuroscience 45, 258-266) and Sunberg et al., 2013 (“Improved CellTherapy Protocols for Parkinson's disease based on differentiationefficiency and safety of hESC-, hiPSC-, and non-human primateiPSC-derived dopaminergic neurons,” Stem Cells. 2013 August;31(8):1548-62).

LRRK2-Specific Anti-Sense Oligonucleotide Treatment.

LRRK2 G2019S exon 41 anti-sense oligonucleotide sequence5′-AGACAGACCTGATCACCTACCTGGT-3′ (SEQ ID NO: 2) and a non-sense controloligo sequence 5′-CCTCTTACCTCAGTTACAATTTATA-3′ (SEQ ID NO: 5) wereadministered to the neuronal culture at DIV 37 and 41. Each time neuronswere treated with 10 μM LRRK2 ASOs using Endo-Porter delivery methodaccording to the manufacturer's protocol (Gene Tools, 4 μl per ml).Depending on the experimental set up, ASOs with a carboxyflourescein tagwere used when the visualization of the ASO in the cell was desired,emitting green-fluorescence at 524.5 nm.

Calcium Imaging

Total Intracellular Calcium Imaging Using Fura2.

iPS-derived neurons were grown on cover slips coated with 15%poly-L-ornithine/laminin in 24 well plates at 50,000 cells/well. Cellcultures were treated with ASOs at DIV 37 and DIV41 as described above.Cells were treated with 0 nM THP or 10 nM THP and imaged 24 hours later(either 7 days post-plating for 0 nM or 8 days post-plating for 10 nMtreatment). iPS-neurons were incubated with 5 μM Fura2-AM (MolecularProbes, F1221) in differentiation medium for 30 minutes at roomtemperature (RT). Cells were imaged at RT in standard external solution(SES, Boston BioProducts, C-3030; 145 mM NaCl, 5 nM KCl, 2 nM CaCl2, 1mM MgCl2, 10 mM Glucose, 10 mM HEPES, pH 7.4±0.15), using a Nikon ElipseTi microscope with a Q-Imaging® CCD camera, at a 10× magnification (PlanFluo objective, NA 0.30). To measure calcium regulation, influx waselicited with application of 50 mM KCl, according to the protocoldescribed in Table 2.

TABLE 2 Live cell calcium imaging KCl application protocol Time Solutionapplied 0-1 minute standard external solution (SES) 1-1 minute 15seconds 50 mM KCl 1 minute 15 seconds-5 minutes SES 5-11 minutes 50 mMKCl 11-13 minutes SES

Intracellular calcium levels were measured by taking the ratio of theFura-2 emission wavelengths; 340 nm and 380 nm, with an exposure time of600 ms and 300 ms respectively, acquiring images every 3 seconds. Toidentify ASO positive cells, one image in the first second was acquiredat 525 nm (Tetramethylrhodamine; TRITC), with an exposure time ofsecond. NIS Elements AR 3.2 imaging software (Nikon) was used to analyzethe acquired images, outlining visually defined cells as regions ofinterest. The Fura-2 340 nm/380 nm ratio fluorescence was measured overtime in the designated regions. The change in fluorescence wasnormalized to the baseline fluorescence prior to the first KClstimulation. Cells with a minimum of 7% change from baseline duringstimulation were considered active cells, and consequently included infurther analyses. Two characteristics of calcium homeostasis weremeasured. Firstly, by analyzing the peak amplitude of intracellularcalcium with the first and second KCl stimulation, representingimmediate calcium influx through calcium channels and cytoplasmiccalcium release from organelles such as the ER. Secondly, the area underthe curve during prolonged depolarization, the second KCl application,was measured. During this state, unbound calcium is cleared bycalcium-binding protein and ER calcium reuptake, allowing calcium levelsreach an equilibrium. Buffering capacity is dependent on inactivation ofcalcium channels and reuptake of calcium by the ER and mitochondria.

ER-calcium imaging using the CEPIA-ER calcium indicator. To measurecalcium levels specifically in the ER, a calcium-measuringorganelle-entrapped protein indicator (CEPIA) was used (Suzuki et al.2014, “Imaging intraorganellar Ca2+ at subcellular resolution usingCEPIA,” Nature Communications, 5:4153). This genetically encoded calciumindicator (GECI) contains an ER specific retention sequence, and has abinding affinity compatible with the sub-millimolar calcium levelsdetected in the ER with an emission wavelength of 511 nm. IPS-derivedneurons were infected with the lentivirus encoding for CEPIA-ER-GFPunder the synapsin promotor 24 hours post-plating, at an MOI of 20.Vehicle treated and 10 nM THP treated neurons where imaged at DIV 43post-plating, 24 hours after the incubation with the vehicle/toxin.Images were acquired at 525 nm, with an exposure time of 800milliseconds for mixed culture iPS-neurons and 1 second for midbrainiPS-neurons, with a second acquisition interval. To measure backgroundbleaching, decay in GFP emission was measured in a non-stimulated trace,for every cell line. Change in fluorescence was corrected for bleachingby dividing the raw measurement point by the average fluorescence ofnon-stimulated cells for every individual acquired time point. ImageJsoftware was used to analyze the acquired images, where regions weredetermined and measured as stated earlier. Baseline ER levels werecalculated based on the mean average of the GFP fluorescence levelsbetween the first 15 and 55 seconds of imaging prior to the KCl. Onlyactive neurons with a minimum of 2% change from baseline during KClstimulation were considered active cells, and consequently included inthe analyses.

Neurite Outgrowth.

After differentiation (DIV 35) EB iPS-derived neurons were replated into96 well plates at 15,000 cells per well for live cell phase imaging ofneurite outgrowth. If needed, on day 2 and day 5 post-plating, neuronswere treated with 10 μM exon 41 ASO (SEQ ID NO:02) or non-target ASO(SEQ ID NO:05) conjugated to carboxyflourescein. Seven days postplating, on DIV 42, neurons were exposed to thapsigargin (THP) toxicityat 0 nM, 1 nM, 10 nM, and 100 nM concentration and the live cell imagingwas started immediately using the IncuCyte® Zoom live imaging system(Essen BioScience) (see FIG. 7). Neurite length per cell body clusterwas measured using the IncuCyte® imaging system. To evaluate the exon 41ASO induced rescue on neurite length, the IncuCyte® ZOOM images wereexported as tiff files and neurites from ASO-carboxyflourescein positiveneurons were manually traced in Fiji ImageJ NeuronJ plugin. Totalneurite length was then corrected for the number of the ASO+ neurons ineach image. Images were randomized and blinded to the investigator.

Statistical Analysis.

Statistical significance was tested using Graphpad Prism 6 software(Graphpad software). 1-way, 2-way ANOVA and student T-test were useddepending on the dataset. Statistical analysis was specified for eachexperiment in the figure legend.

Other Phenotypic Assays:

Mitochondrial Dysfunction Assays.

It was previously shown that human LRRK2 G2019S iPSC-derived neurons andhuman fibroblasts exhibit increased vulnerability to PD associated cellstressors and modified mitochondrial dynamics, which can be rescued byLRRK2 inhibitors (Cooper et al., 2012, “Pharmacological Rescue ofMitochondrial Deficits in iPSC-Derived Neural Cells from Patients withFamilial Parkinson's Disease” Sci Transl Med. 2012, 4; 4(141):141ra90;Smidt et al., “Fibroblast Biomarkers of sporadic parkinson's disease andLRRK2 kinase inhibition,” Mol Neurobiol. 2016 October; 53(8):5161-77).

Nitric Oxide and Super Oxide Live Cell Imaging.

DAF-FM is a cell permeant molecule, which upon reaction with nitricoxide (NO) forms a fluorescent benzotriazole, emitting greenfluorescence at 515 nm (Molecular Probes). On DIV 43, 24 hours postvalinomycin treatment at 0 μM (vehicle control), 2 μM, and 10 μMvalinomycin (Sigma, v0627) dissolved in DMSO (Sigma, D2650-5X5ML), EBneurons were labeled with DAF-FM fluorescent probe (Molecular Probes,D-23844) at 5 μM in HBSS containing calcium and magnesium (LifeTechnologies, 14025-092) according to the manufacturer's protocol. Thecells were immediately imaged using the IncuCyte® Zoom live imagingsystem and DAF-FM green fluorescence intensity was quantified.

MitoSOX™ is a molecule targeting mitochondria throughtriphenylphosphonium (Molecular Probes). MitoSOX™ is oxidized by superoxide, consequently emitting red fluorescence at 580 nm. 24 hours postvalinomycin treatment EB neurons were labeled with the MitoSOX™(Molecular Probes, M36008) fluorescent probe at 7.5 μM in HBSS for 30minutes at 37° C. After three washes with HBSS, the cells wereimmediately imaged using the IncuCyte® Zoom live imaging system andMitoSox red fluorescence was quantified.

Mitochondrial Labeling with MitoTracker®.

At DIV 42 mitochondria of EB neurons were labeled with MitoTracker® Red(Molecular Probes, M-22425) at 250 nM concentration for 45 minutes at37° C. Cells were then washed with fresh neural differentiation mediaand immediately imaged using the IncuCyte® Zoom live imaging system.Phase contrast as well as red fluorescence images were acquired every 3hours for 4 days, starting 2 hours before the valinomycin treatment.

Mitophagy Assay.

ATP3-Rosella plasmid was acquired from and previously characterized byRosado et al., 2008, (“Rosella: A fluorescent pH-biosensor for reportingvacuolar turnover of cytosol and organelles in yeast.” Autophagy 4,205-213). The ATP3-Rosella sequence was inserted into a lentivirusbackbone with a CMV promotor. HEK 293T cells were transfected with 0.4μg of the lenti-Rosella plasmid DNA using polyethylenimine (PEI). 48hours post transfection cells were treated with 1 μM rotenone and 20 nMrapamycin and imaged every 3 hours for 3 days using the IncuCyte® Zoomlive imaging system. Phase contrast, green fluorescence, and redfluorescence images were acquired.

Neurons differentiated using the EB protocol were infected with thelentivirus expressing the rosella bioprobe at MOI 10 on DIV 36.Valinomycin (2 μM) and Rotenone (1 μM) toxicities were applied toneurons on DIV 42, after which cells were imaged every 3 hours for 3days using the IncuCyte® Zoom live imaging system. Phase contrast, greenfluorescence, and red fluorescence images were acquired. The ratio ofgreen/red fluorescence intensity was quantified.

Fibroblasts from healthy subject and Parkinson disease patients wereinfected with lentivirus expressing the rosella bioprobe at MOI 75 4hpost-plating into 12 well plates with 60,000 cells per well. Two dayslater, cells were replated into 96 well plates with 2,000 cells perwell. On the following day, DIV4, fibroblasts were transfected with ASOstargeting exon 41 (SEQ ID NO:02), exon 31 (SEQ ID NO:01), exon 2 (SEQ IDNO:07), and control non-target ASO (SEQ ID NO:05). Two days later, livecell imaging of the mitophagic flux was performed using the IncuCyte®Zoom live imaging system. Phase contrast, green fluorescence, and redfluorescence images were acquired. The ratio of green/red fluorescenceintensity was quantified.

Endoplasmic Reticulum Stress Response.

After differentiation (DIV 35) EB iPSC-derived neurons were replatedinto 96 well plates at 15,000 cells per well. Five days post plating atDIV40, neurons were infected with ER stress Cignal™ Lenti Reporter AssayERSE and ATF6 (CLS-9032L-8, CLS-6031L-8, Qiagen), at MOHO. Seven dayspost plating, on DIV 42, neurons were exposed to 24 hours incubationwith thapsigargin (THP) at 0 nM, 1 nM, 10 nM, and 100 nM concentration.One day later luciferase activity was measured using the Dual-Glo®Luciferase Assay System (E2940, Promega) (see FIG. 13B and FIG. 13C).

For gene expression analysis, RNA was isolated from neurons cultured ina 96 well plates seeded at a density of 15,000 cells/well at DIV43 24hours post THP treatment, using the QIAshredder™ spin columns and theRNeasy® spin column method (Qiagen, 74104). cDNA synthesis was performedaccording to the QuantiTect® Reverse Transcription procedure (Qiagen,205311); QuanTitect® primer assays were used for detection of the ATF6A,ATF6B and YY1 genes (QT00083370, QT00009380, QT00052738, Qiagen), usingSybrGreen detection method (Applied Biosystems, 4367659) and StepOnePlusReal-Time PCR system (Applied Biosystems) according to the manufacturerprotocol. Gene expression levels were normalized to GAPDH (IDT)expression.

Cell Viability Assay:

In vitro toxicity was determined by lactate dehydrogenase (LDH) assay(Roche) to measure the conversion of a tetrazolium substrate by LDHenzyme released through the cell plasma membrane during cell death.Fibroblasts were placed in a clear, flat-bottomed 96-well plate at adensity of 2,000 cells per well. One day post-plating, cells weretransfected with exon 41 ASO (SEQ ID NO:02) at 1 μM, 5 μM and 15 μM andnon-target ASO (SEQ ID NO:05) at 15 μM. Forty-eight hours later, cellswere incubated at 37° C. with 40 μM valinomycin for 24 hours. Thesubstrate and catalyst were applied according to the manufacturer'sinstructions. The LDH-based colorimetric change was analyzed accordingto the manufacturer's instructions using the Spectra Max Plus 384spectrophotometer and Soft Max Pro 5.4.4 software. The optical densityof each line was subtracted from a lysed control for that same line andsamples were run in three technical replicates.

LRRK2 Protein Analysis in Human Fibroblasts:

Healthy subject and Parkinson's disease patient fibroblasts were platedat 500,000 cells into 24 well plate. One day post plating, fibroblastswere transfected with exon 41 (SEQ ID NO:02), exon 31 (SEQ ID NO:01),exon 2 (SEQ ID NO:07), and non-target ASO (SEQ ID NO:05) withEndo-Porter as described above. Two days later, at DIV4, cells werelysed using ice-cold RIPA lysis buffer (Thermo Fisher Scientific,PI-89900) containing Halt Protease & Phosphatase Inhibitor Cocktail(1:100, Thermo Fisher Scientific, 1861284) and 0.5M EDTA Solution(1:100, Thermo Fisher Scientific, 1861283). Samples were sonicated andprotein content was quantified using the BCA Protein Assay Kit (BCAAssay, Thermo Scientific, 23227). Samples were prepared to aconcentration of 40 μg in distilled water and Pierce™ Lane MarkerReducing Sample Buffer (Thermo Fisher Scientific, 39000) and boiled for5 minutes at 100° C. Protein samples were then separated in 4-20%Tris-HCL (345-0032) Criterion precast gels (Bio-rad) and transferredonto Trans-Blot® Turbo™ 0.2 μm PVDF membranes (Bio-rad, 170-4157) byapplying an electrical charge of 21 V and 2.5 A for 7 minutes. Membraneswere incubated in blocking buffer (TBS, 0.1% (v/v) Tween-20 (Fisher,BP337-500) and 5% blotting grade blocker (Bio-rad, 1706404)) for 1 hourat room-temperature. Membranes were then cut and incubated with Ser935LRRK2 UDD2 antibody (1:250, Abcam) overnight on a shaker at 4° C. Thenext day, blots were incubated with a goat-anti-rabbit (1:5000, Bio-rad,170-6515) conjugated secondary antibody for 1 hour at room-temperature.Blots were developed using the Advansta WesternBright Sirius kit(Advansta, Cat. # K-12043-D10) and imaged using ChemiDoc™ XRS+ system(Bio-rad). After imaging, blots were stripped with stripping buffer,block in 5% milk-TBST solution and incubated with LRRK2 (100-500) UDD3antibody (1:250, MRC, University of Dundee, Monoclonal Rabbit 30-12) oranti-GAPDH antibody (1:10,000 AB2302, Millipore) in blocking buffer overthe weekend on a shaker at 4° C. Next, blots were incubated with agoat-anti-rabbit (1:5000, Bio-rad, 170-6515) or anti-chicken IgY HRP(1:5000, G135A, Promega) conjugated secondary antibodies for 1 hour atroom-temperature. Blots were developed using the Advansta WesternBrightSirius kit (Advansta, Cat. # K-12043-D10) and imaged using ChemiDoc™XRS+ system (Bio-rad). Blot analysis was performed using Image Lab™analysis software.

LRRK2 Protein Analysis in Human iPSC-Derived Neurons:

Post differentiation at DIV 35, iPSC-derived neurons were plated onto 12well plates at 500,000 cells per well. Neurons were treated 2× with exon41 (SEQ ID NO:02), exon 31 (SEQ ID NO:01), and non-target ASOs (SEQ IDNO:05) at 10 μM concentration with Endo-Porter as described above. Onday 7 post-plating, neurons were lysed using ice-cold RIPA lysis buffer(Thermo Fisher Scientific, PI-89900) containing Halt Protease &Phosphatase Inhibitor Cocktail (1:100, Thermo Fisher Scientific,1861284) and 0.5M EDTA Solution, (1:100, Thermo Fisher Scientific,1861283). Samples were sonicated and protein content was quantifiedusing the BCA Protein Assay Kit (BCA Assay, Thermo Scientific, 23227).Samples were prepared to a concentration of 40 μg in distilled water andPierce™ Lane Marker Reducing Sample Buffer (Thermo Fisher Scientific,39000) and boiled for 5 minutes at 100° C. Protein samples were thenseparated in 4-20% Tris-HCL (345-0032) Criterion precast gels (Bio-rad)and transferred onto Trans-Blot® Turbo™ 0.2 μm PVDF membranes (Bio-rad,170-4157) by applying an electrical charge of 21 V and 2.5 A for 7minutes. Membranes were incubated in blocking buffer (TBS, 0.1% (v/v)Tween-20 (Fisher, BP337-500) and 5% blotting grade blocker (Bio-rad,1706404)) for 1 hour at room-temperature. Membranes were then cut andincubated with LRRK2 (100-500) UDD3 antibody (1:250, MRC, University ofDundee, Monoclonal Rabbit 30-12) or anti-GAPDH antibody (1:10,000AB2302, Millipore) in blocking buffer over the weekend on a shaker at 4°C. Next, blots were incubated with goat-anti-rabbit (1:5000, Bio-rad,170-6515) or anti-chicken IgY HRP (1:5000, G135A, Promega) conjugatedsecondary antibodies for 1 hour at room-temperature. Blots weredeveloped using the Advansta WesternBright Sirius kit (Advansta, Cat. #K-12043-D10) and imaged using ChemiDoc™ XRS+ system (Bio-rad). Blotanalysis was performed using Image Lab™ analysis software.

Example 1: Antisense Oligonucleotides Induce Skipping of Exon 41 andExon 31 in Human LRRK2 Gene in Human iPS-Derived Neurons

ASO 41-1 (see Table 3; SEQ ID NO:02) was tested in the human iPS-derivedneural cell line. ASO 41-1 (10 μM final concentration, one treatment atDIV41) was transfected into cells using Endo-Porter (GeneTools)according to manufacturer's protocol. FIGS. 3A and 3B demonstrate thatASO 41-1 induces skipping of targeted exon 41 of LRRK2 with or withoutthe G2019S mutation (also see FIG. 38A and FIG. 38C).

ASO 41-1 and a non-sense control were tested in human iPS-derivedneurons (ASO41-1 is 5′-AGACAGACCTGATCACCTACCTGGT-3′; SEQ ID NO: 2) and anon-sense control oligonucleotide sequence (Non-sense control is5′-CCTCTTACCTCAGTTACAATTTATA-3′; SEQ ID NO: 5) either once (5 days postdifferentiation, DIV37) or two times (2 and 5 days post end of thedifferentiation (DIV37 and DIV41)). Each time neurons were treated with10 μM LRRK2 ASOs using Endo-Porter delivery method according to themanufacturer's protocol (Gene Tools, 4 μl per 1 mL solution). Exonskipping efficiency was also assessed by comparing between the non-tagand the carboxyflourescein-3′ tag ASO sequences. RNA was collected 48hours post the second transfection (DIV43). LRRK2 exon 41 skipping wasinduced by ASO 41-1 in iPS-derived neurons derived from healthy subjectcontrols (CTRL 10A and 21.31) or PD patients carrying LRRK2 G2019Smutation (G2019S 29F and PD28) (see FIG. 4A and FIG. 4B). FIG. 39 showsexon 31 skipping by ASO#5 (SEQ ID NO:01) in iPSC-derived human neuronseither from a control healthy subject (N=3) or PD patient carrying theLRRK2 R1441C mutation (N=1). LRRK2 protein levels were also decreased iniPS-derived neurons by ASOs targeting either exon 41 or exon 31 (seeFIG. 38B and FIG. 40)

TABLE 3 Antisense oligonucleotide targeting LRRK2 induceexon 41 skipping. Target % SEQ ID Name Exon Sequence skipped * NO. 41-141 AGACAGACCTGATCACCTACCTGGT 62 SEQ ID NO. 2 * percent of the mRNAtranscripts that skip out the targeted exon

Example 2: Antisense Oligonucleotides Successfully Reduce Full-LengthLRRK2 Expression of LRRK2 Exon 31 and 41 in Fibroblast Cells fromParkinson's Patients

Various ASOs (see Table 4; SEQ ID NOs: 1 and 2) were tested in humanfibroblast cells from one healthy subject control and two patients withParkinson's disease one carrying the R1441C mutation and one carryingthe G2019S mutation. ASOs (7.5 μM final concentration of either ASO 31-1or ASO 41-1) were transfected into cells using Endo-Porter (GeneTools)according to manufacturer's protocol. FIG. 2 demonstrates that ASOs 31-1and 41-1 induce skipping of targeted exons 31 and 41 in human LRRK2,respectively.

TABLE 4 Antisense oligonucleotides targeting LRRK2 induce exon skippingTarget % SEQ ID Name Exon Sequence skipped * NO. 31-1 31CTACCAGCCTACCATGTTACCTTGA 40 SEQ ID NO. 1 41-1 41AGACAGACCTGATCACCTACCTGGT 31 SEQ ID NO. 2 * percent of the mRNAtranscripts that skip out the targeted exon

The results provided in the examples and figures above demonstratethat: 1) human neurons carrying LRRK2 G2019S mutation show a decreasedlevel of the UPR response after ER calcium depletion indicating a lowercapacity to adapt to the ER stress; 2) LRRK2 G2019S iPS-derived neuronshave a decreased neurite integrity during ER stress; 3) intracellularcalcium homeostasis is dysregulated in iPS-derived PD patient neuronscarrying LRRK2 G2019S mutation during ER stress; and 4) ER calciumlevels, intracellular calcium uptake (during ER stress) and neuriteintegrity (during ER stress) can be restored in LRRK2 G2019S neuronsupon antisense oligonucleotide treatment targeting the G2019S mutation.

Example 3: Antisense Oligonucleotides Induced Exon Skipping inFibroblast Cells from Parkinson's Patients

Various ASOs (see Table 5) were tested in human fibroblast cells fromone healthy subject control and two patients with Parkinson's diseaseone carrying the R1441C mutation and one carrying the G2019S mutation.ASOs (at concentrations of 1.0 μM, 5 μM, 7.5 μM, 15 μM, 22.5 μM, or 45μM) were transfected into cells using Endo-Porter (GeneTools) accordingto manufacturer's protocol. FIGS. 16-26 and 38-39 demonstrate that theASOs disclosed in Table 5 induce skipping of targeted exons in humanLRRK2.

TABLE 5 Antisense oligonucleotides targeting LRRK2. ASO Name ExonSequence (SEQ ID NO.) ASO#23 or exon 4 ATACACATATTACCTGAAGTTAGGA ASO 4-1(SEQ ID NO: 06) ASO#45 or exon 2 AGTGAAAACAATGCCTTTACCTGCT ASO 2-1(SEQ ID NO: 07) ASO#46 exon 41 GGTATCTGCCAGAAAATGCACAGGA 41-2(SEQ ID NO: 08) ASO#284 exon 41 -   AATGCtGTAGTCAGCAATCTTTGCA G2019  (SEQ ID NO: 09) mutant specific ASO#6 or exon 41AGACAGACCTGATCACCTACCTGGT ASO 41-1 (SEQ ID NO: 02) ASO#5 or exon 31CTACCAGCCTACCATGTTACCTTGA ASO 31-1 (SEQ ID NO: 01)

Example 4: Antisense Oligonucleotides Successfully Reduce Full-LengthLRRK2 Expression in Fibroblast Cells from Parkinson's Patients

Various ASOs (see Table 5) were tested in human fibroblast cells fromthree healthy subject control and three patients with Parkinson'sdisease carrying either the R1441C mutation or the G2019S mutation. ASOs(at concentrations of 1.0 μM, 5 μM and 15 μM) were transfected intocells using Endo-Porter (GeneTools) according to manufacturer'sprotocol. FIGS. 31-37 demonstrate that the ASOs disclosed in Table 5induce skipping of targeted exons in human LRRK2 and can reduce LRRK2protein levels in the patient derived cells.

Example 5: Mutant LRRK2 Dependent Dysregulation of Mitophagy

Various ASOs (see Table 5) were tested in human fibroblast cells fromfive healthy subject control and five patients with Parkinson's diseaseeither carrying the R1441C mutation or the G2019S mutation. ASOs (atconcentrations of 1.0 μM, or 5 μM) were transfected into cells usingEndo-Porter (GeneTools) according to manufacturer's protocol. FIGS. 25,and 27-30 demonstrate that the ASOs disclosed in Table 5 induce skippingof targeted exons in human LRRK2 and can successfully rescue ofmitophagic flux in LRRK2 G2019C or LRRK2 R1441C fibroblasts upon LRRK2exon skipping.

Example 6: Exon 41 LRRK2 ASO Skipping Rescues Neurite Outgrowth THPInduced Collapse in LRRK2 G2019S Neurons

After differentiation iPS-derived neurons were replated for live cellphase imaging of neurite outgrowth. Seven days post plating, neuronswere exposed to thapsigargin (THP) toxicity at 0 nM, 10 nM, and 100 nMconcentrations and the live cell imaging was started immediately usingthe IncuCyte® Zoom live imaging system (Essen BioScience). Neuritelength per cell body cluster was measured using the IncuCyte® imagingsystem or ImageJ software. FIG. 7 demonstrates that exon 41 LRRK2 ASOskipping rescues neurite outgrowth in LRRK2 G2019S neurons.

Example 7: Exon 41 LRRK2 ASO Skipping and Nitric Oxide Levels in LRRK2G2019S Neurons

iPS-derived neurons carrying the LRRK2 G2019S mutation show asignificant increase in nitric oxide levels after mitochondrial membranedepolarization induced by 24 hours low dose valinomycin treatment. FIG.8C demonstrates that exon 41 LRRK2 ASO skipping appears to prevent anincrease in nitric oxide levels in LRRK2 G2019S neurons followingvalinomycin treatment.

Having described the invention in detail and by reference to specificembodiments thereof, it will be apparent that modifications andvariations are possible without departing from the scope of theinvention defined in the appended claims. More specifically, althoughsome aspects of the present invention are identified herein asparticularly advantageous, it is contemplated that the present inventionis not necessarily limited to these particular aspects of the invention.

What is claimed is:
 1. A method of modulating splicing or expression ofa LRRK2 transcript in a cell comprising contacting the cell with atleast one compound comprising a modified oligonucleotide having 8 to 30linked nucleosides and having a nucleobase sequence comprising acomplementary region, wherein the complementary region comprises atleast 8 contiguous nucleobases complementary to an equal-length portionof a target region of a Leucine-Rich-Repeat-Kinase (LRRK2) transcript,wherein the modified oligonucleotide has a nucleobase sequence as setforth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:7, or SEQ IDNO:8.
 2. The method of claim 1, wherein the cell is in vitro or in vivo.3. The method of claim 1, wherein the cell is in vivo in an animal andthe animal is a human.
 4. The method of claim 3, wherein the modifiedoligonucleotide is administered to the human by intrathecal injection,intracerebroventricular injection, inhalation, parenteral injection orinfusion, oral, subcutaneous or intramuscular injection, buccal,transdermal, transmucosal, or topical.
 5. The method of claim 1, whereinthe target region of the modified oligonucleotide comprises at least aportion of exon 2, exon 4, exon 31, or exon 41 of the LRRK2 transcript.6. The method of claim 1, wherein the modified oligonucleotide comprisesa nucleobase sequence having 20-30 linked nucleoside and having thenucleobase sequence as set forth in SEQ ID NO:9.
 7. The method of claim1, wherein the modified oligonucleotide comprises at least one modifiednucleoside selected from a modified sugar moiety, a 2′-substituted sugarmoiety, a 2′OME, a 2′F, a 2′-MOE, a bicyclic sugar moiety, a LNA, a cEt,a sugar surrogate, a morpholino, or a modified morpholino.
 8. The methodof claim 1, wherein the complementary region of the modifiedoligonucleotide comprises 10-25 contiguous nucleobases.
 9. The method ofclaim 1, wherein the compound further comprises a pharmaceuticallyacceptable carrier or diluent.
 10. The method of claim 4, wherein themodified oligonucleotide is administered by intrathecal injection or byintracerebroventricular injection.
 11. The method of claim 1, whereinthe modified oligonucleotide consists of the nucleobase sequence of SEQID NO:7.
 12. The method of claim 7, wherein the modified nucleoside is amorpholino.
 13. The method of claim 3, wherein the human has one or moresymptoms associated with Parkinson's disease.
 14. The method of claim 1,wherein the modified oligonucleotide consists of the nucleobase sequenceof SEQ ID NO:6.
 15. The method of claim 1, wherein the modifiedoligonucleotide consists of the nucleobase sequence of SEQ ID NO:8. 16.The method of claim 6, wherein the modified oligonucleotide consists ofthe nucleobase sequence of SEQ ID NO:9.